Extranuclear RNA splicing in neuronal dendrites

ABSTRACT

The present invention relates to methods of synaptic network remodeling by means of extranuclear RNA splicing. The present invention also provides methods of extranuclear RNA splicing, and methods of protein translation based on extranuclear RNA splicing.

BACKGROUND OF THE INVENTION

In a living cell, such as a neuron, from the moment a primary RNAtranscript is complete to the actual expression of the protein encodedby the transcript, multiple cellular events and mechanisms occur,including pre-RNA splicing, RNA editing, shuttling of the mRNA betweenthe nucleus and the cytoplasm, and processes that ensure the stabilityand translational control of the trafficked mRNAs. Each of these eventsprovides opportunities for the cell to regulate gene expression at theRNA level.

Each neuron is comprised of a nucleus within a body, or soma, a longfiber called the axon, and a varying number of branching fibers calleddendrites, which extend out to other neurons. A single neuron can makenumerous contacts with other neurons and tissues. For example, every newthought process is handled by a new set of synaptic connections. Memoryitself is a set of synaptic connections engraved in the network ofneurons.

Dendrites are specialized extensions of the neuronal soma that containcomponents of the cellular machinery involved in RNA and proteinmetabolism. A subset of mRNAs are trafficked to dendrites through theirassociation with RNA binding proteins (RBPs). Some of these RBPsfunction in the nucleus as mediators of pre-RNA splicing.

The functional properties of neurites, including dendrites and axons,have been extensively examined since the discovery of protein syntheticmachinery in dendrites, including ribosomes and membranous constituentsof the endoplasmic reticulum and golgi apparatus (Bodian, 1965, Proc.Natl. Acad. Sci. U.S.A., 53:418-425; Steward et al., 1983, Res.58:131-6; Torre et al., 1996, J. Neurosci. 16:5967-78; Gardiol et al.,1999, J. Neurosci. 19:168-79). Increasingly, more detailed molecularanalyses of dendrites have shown that a subset of cellular RNAs aretransported into dendrites where they can be translated into protein atspecialized areas following synaptic stimulation (Aakalu et al., 2001,Neuron 30:489-502; Bassell et al., 1998, J. Neurosci. 18:251-65; Crinoet al., 1996, Neuron 17:1173-87; Huber et al., 2000, Science 288:1254-7;Job et al., 2001, Proc. Natl. Acad. Sci. U.S.A 98:13037-42; Martin etal., 1997, Cell, 91:927-38). In the cytoplasm, the intracellulartransport, stability, and translation of RNA are regulated by RNAbinding proteins (RBPs) (Spirin et al., 1979, Mol. Biol. Rep. 5:53-57).RBP-RNA interactions typically occur through conserved motifs in RBPsthat associate with cis acting sequences or secondary structures in RNA.

Recently, other RBPs thought to function only in the nucleus have alsobeen localized in the cytoplasm. These include RNA editing enzymes (e.g.double stranded RNA adenosine deaminase) (Strehblow et al., 2002, Mol.Biol. Cell, 13:3822-35) as well as some of the highly conservedconstituents of the spiceosome (e.g. the survival of motor neuron proton(Fan et al., 2002, Hum. Mol. Genet. 11: 1605-14) and a variety ofheterogeneous nuclear ribonucleoproteins (hnRNPs) (Pinol-Roma, 1997,Semin. Cell Dev. Biol. 8:57-63). Some auxiliary components of thespliceosome, such as the splicing factor SAM68, are present within thesomatodendritric compartment of neurons as well (Staley et al., 1998,Cell 92:315-26; Jurica et al., 2003, Mol. Cell 12:5-14; Grange et al.,2004, J. Neurosci. Res., 75:654-66). The presence of these proteins in anon-nuclear compartment suggests that they either serve a uniquefunctional role outside of the nucleus or their known functionalactivity can occur within this subcellular compartment.

The spliceosome, which catalyses the ATP-dependent removal of intronsfrom nuclear pre-RNA, is a multi-megadalton complex of proteins andsmall nuclear RNAs (snRNA) (Staley et al., 1998, Cell 92:315-26; Juricaet al., 2003, Mol. Cell 12:5-14). Even in the nucleus, the distributionof pre-RNA splicing factors is not uniform. Rather, within discretesites of concentration and lower levels of factors diffusely dispersedthroughout the nucleoplasm, speckles (splicing factor compartments) canbe readily identified with an antibody against the spliceosome assemblyfactor SC-35 (Lamond et al., 2003, Nat. Rev. Mol. Cell Biol. 4:605-12).

Despite the existing knowledge of these nuclear factors in thecytoplasm, the current state of the art does not definitively attributefunction or role to the presence of RBPs and spliceosome components inthe cytoplasm. A greater understanding of the regulation, metabolism andgrowth of cells will enable more accurate and more useful control andmanipulation of cells. The development of such tools can enable moreprecise, targeted therapies and treatments of all mammals, and inparticular, of humans. Therefore, there exists a need for a betterunderstanding of the function and role of RBPs and spliceosomecomponents in the cytoplasm in order to facilitate the controlledmanipulation of cells. The present invention addresses and meets theseneeds.

SUMMARY OF THE INVENTION

The present invention features a method of remodeling a dendritecomprising the steps of transfecting a dendrite with an RNA comprisingat least one intron, wherein the dendrite comprises at least onecomponent of a spliceosome and further wherein the component of aspliceosome is capable of splicing an RNA. The method also includes thestep of allowing the RNA comprising at least one intron to be spliced bythe spliceosome components and allowing the spliced RNA to be translatedin the dendrite, wherein the dendrite is thereby remodeled as aconsequence of the translation.

The invention also features a method of remodeling a dendriteinteraction comprising the steps of transfecting a dendrite with an RNAcomprising at least one intron, wherein the dendrite comprises at leastone component of a spliceosome and further wherein the component of aspliceosome is capable of splicing an RNA. The method also includes thestep of allowing the RNA comprising at least one intron to be spliced bythe spliceosome components and allowing the spliced RNA to be translatedin the dendrite, wherein the dendrite interaction is thereby remodeledas a consequence of the translation.

The present invention further features a method of remodeling a synapticnetwork comprising interaction with at least one dendrite comprising thesteps of transfecting a dendrite with an RNA comprising at least oneintron, wherein the dendrite comprises at least one component of aspliceosome and further wherein the component of a spliceosome iscapable of splicing an RNA. The method also includes the step ofallowing the RNA comprising at least one intron to be spliced by thespliceosome components and allowing the spliced RNA to be translated inthe dendrite, wherein the synaptic network is thereby remodeled as aconsequence of the translation.

In one aspect of the invention, a dendrite is a component of a neuron.In another aspect, a dendrite is an isolated dendrite.

The invention also provides a method of splicing an RNA comprising thesteps of providing an isolated dendrite comprising at least onecomponent of a spliceosome, wherein the component is capable of splicingan RNA, and transfecting the dendrite with an RNA comprising at leastone intron, wherein the RNA comprising at least one intron is spliced bythe spliceosome components.

The invention further provides a method of splicing an RNA comprisingthe steps of providing an isolated cell comprising at least onecomponent of a spliceosome in the cytoplasm, wherein the component iscapable of splicing an RNA, and transfecting a cell with an RNAcomprising at least one intron, wherein the RNA comprising at least oneintron is spliced by the spliceosome components in the cytoplasm.

In one aspect of the invention, the RNA splicing donor/acceptor pairsare selected from the group consisting of canonical, atypical andcryptic. In another aspect of the invention, an RNA comprising at leastone intron is a pre-RNA. In another aspect, a pre-RNA is a pre-mRNA.

In one embodiment of the invention, an RNA comprising at least oneintron is derived from a nucleic acid comprising a construct selectedfrom the group consisting of pEGFP-N1, pDsRed-N1, SF1/mBBP-GFP,U2AF65-GFP, a GFP construct, a DsRed construct, a histone 2B-YFPconstruct.

In another embodiment of the invention, at least one spliceosomecomponent is selected from the group consisting of Y14, Magoh, RNPS1,SC-35, SF2, U2AF65, Smith antigen, pan-SR antigen, U1 snRNP, U2 snRNP,U4 snRNP, U5 snRNP, U6 snRNP.

The invention also features a method of translating an RNA, comprisingthe steps of providing an isolated dendrite comprising at least onecomponent of a spliceosome, wherein the component is capable of splicingan RNA, and transfecting the dendrite with an RNA comprising at leastone intron, wherein the RNA is spliced by the spliceosome components.

The invention features a method of translating an RNA, comprising thesteps of providing an isolated cell comprising at least one component ofa spliceosome in the cytoplasm, wherein the component is capable ofsplicing an RNA, and transfecting the cell with an RNA comprising atleast one intron, wherein the RNA is spliced by the spliceosomecomponents in the cytoplasm. The method further includes translation ofthe spliced RNA.

The present invention also provides a method of splicing an RNA,comprising the steps of providing an isolated synaptoneurosomecomprising at least one component of a spliceosome, wherein thecomponent is capable of splicing an RNA, contacting saidsynaptoneurosome with an RNA comprising at least one intron, wherein theRNA comprising at least one intron is spliced by the spliceosomecomponents.

The invention also features a method of translating an RNA, comprisingthe steps of providing an isolated synaptoneurosome comprising at leastone component of a spliceosome, wherein the component is capable ofsplicing an RNA, contacting the synaptoneurosome with an RNA comprisingat least one intron, wherein the RNA is spliced by the spliceosomecomponents, and contacting the spliced RNA with a composition capable oftranslating an RNA under conditions suitable for translating an RNA.

BRIEF DESCRIPTION OF THE DRAWINGS

For the purpose of illustrating the invention, there are depicted in thedrawings certain embodiments of the invention. However, the invention isnot limited to the precise arrangements and instrumentalities of theembodiments depicted in the drawings.

FIG. 1 is a chart depicting sequences of dendritically-spliced andsynaptoneurosome-spliced RNAs.

FIG. 2, comprising FIGS. 2A through 2L, is a series of imagesdemonstrating coexistence of pre-spliceosome proteins in neuronaldendrites. FIGS. 2A through 2C are images of cultured hippocampalneurons labeled with Qdots 525/605 for detection of SC-35, U2AF andmerged of FIGS. 2A and 2B, respectively. FIGS. 2D through 2F are imagesof cultured hippocampal neurons labeled with Qdots 525/605 for detectionof SC-35, Sm antigen and merged of FIGS. 2D and 2E, respectively. FIGS.2G through 2I are images of cultured hippocampal neurons labeled withQdots 525/605 for detection of SC-35, SF2, and merged of FIGS. 2G and2H, respectively. FIGS. 2J through 2L are images of cultured hippocampalneurons labeled with Qdots 525/605 for detection of SR, SF2, and mergedof FIGS. 2J and 2K, respectively. MAP2 protein was detected in allsamples via Cy5 immuno-detection and is shown in the inset panel in FIG.2A. Qdot secondary antibodies alone without primary antibodies (seeinset in FIGS. 2G and 2H) do not produce an observable signal in thesecells.

FIG. 3, comprising FIGS. 3A through 3F, is a series of imagesdemonstrating detection of U1 snRNA in dendrites. Neurons were subjectedto in situ hybridization (ISH) with antisense digoxigenin-labeled U1 RNA(FIG. 3A) and a competition control where an excess of unlabelled U1antisense RNA was hybridized to the section followed by washing andaddition of digoxigenin-labeled RNA (FIG. 3B). Control ISH useddigoxygeninlabeled GAD65 antisense RNA (FIG. 3E). Hybridized RNA wasdetected by alkaline phosphatase conjugated anti-digoxigenin antibody inthe presence of NBT/BCIP. A strong nuclear presence of U1 RNA along withmoderate staining within several dendrites was observed. Without wishingto be bound by any particular theory, background nuclear staining inFIG. 3B is attributed to hybridization to U1 genes and pseudogenes.FIGS. 3C and 3D show MAP2 immunofluorescence of antisense andcompetition control U1 in situ hybridizations, respectively, in order toidentify the dendrites on these neurons. FIG. 3E depicts that GAD65 RNAis predominantly localized to the cell soma. There was little to noobservable GAD65 ISH signal in the neuronal processes (as observed byphase contrast in FIG. 3F). The bright halo surrounding the neuron inphase-contrast (FIG. 3F) is a characteristic of healthy neurons whetherlive or fixed.

FIG. 4, comprising FIGS. 4A through 4O, is a series of imagesdemonstrating distribution of selected components of spliceosomalsubcomplexes A, B*, and C. Olympus Fluoview FV1000 images of primaryhippocampal cells show the subcellular localization of the selectedcomponents of each spliceosome subcomplex. For subcomplex A, comprisingFIGS. 4A through 4D, SF1/mBBP-GFP (FIG. 4A) and U2AF65-GFP (FIG. 4C)were used. Lower magnification whole cell views were stained for MAP2with a Cy3 secondary antibody (FIGS. 4A-1 and 4C-1). A hatched boxdenotes the area highlighted by higher magnification views. Highermagnification of SFI/mBBP-GFP (FIGS. 4B and 4B-1) and U2AF65-GFP (FIGS.4D and 4D-1) and their corresponding MAP2 immunofluorescence illustratethe presence of small puncta expressed throughout the dendrite. Forsubcomplex B*, immunofluorescence of anti-PSF monoclonal antibody inwhole cell (FIG. 4E-1) and higher magnification view (FIG. 4F-1) areshown. Corresponding MAP2 immunofluorescence is shown using Alexa 488secondary antibody for each of these views (FIGS. 4E and 4F). Forsubcomplex C, immunofluorescence of anti-Aly/REF monoclonal antibodywith Cy3 secondary antibody in low (FIG. 4G-1) and high (FIG. 4H-1)magnification views is shown. Corresponding MAP2 immunofluorescence isshown using Alexa 488 (FIGS. 4G and 4H). GFP fusion constructs forMagoh, UAP56, or Y14 were used. Whole cell (FIGS. 4I, 4L, and 4N) orhigher magnification of dendritic fields (FIGS. 4, 4J, 4M, and 4O) showthe diffusely and more concentrated puncta within the dendroplasm. MAP2immunofluorescence was visible in the red channel in both low (FIGS.41-1, 4L-1, and 4N-1) and high magnification (FIGS. 4M-1 and 4O-1). FIG.4K depicts a whole cell view of a glial cell tranfected with theMagoh-GFP construct. It was observed that Magoh-GFP expression andU2AF65-GFP show low levels of GFP expression with a readily visiblenuclear distribution and diffused GFP fluorescence throughout the glialcytoplasm.

FIG. 5, comprising FIGS. 5A and 5B, demonstrates detection of CDCpre-RNA splicing in an Sm-antigen positive dendrite. FIG. 5A depicts aninset of an area harvested for detection of spliced products. The yellowappearance of the neuronal nuclei is attributed to blending of the greennuclear fluorescent immunostaining of Sm protein and the red cytoplasmicfluorescent immunostaining of the MAP2 protein. FIG. 5B is a schematicof a methodology for CDC pre-RNA splicing. Briefly, after RNAtransfection, in situ transcription (IST) of CDC RNA andimmunofluorescence (IMF) detection of Sm granules was performed. Smproteins positive dendrites were harvested and their nucleic acidcomponents were amplified for spliced CDC RNA content. These PCRamplicons were cloned and sequenced. An intron/exon sequence was derivedfrom the dendrite harvested in FIG. 5A using this methodology.

FIG. 6 is a image depicting a western blot detection of splicingproteins in synaptoneurosomes. Homogenates of whole brain (WB) andsynaptoneurosomes (SN) were probed with the corresponding antibody.Numbers on the left depict the relative molecular weight of the proteinbands (KDa). The tubulin signal is included at the bottom of theautoradiograms to demonstrate that the same amount of protein was loadedfor the whole brain and synaptoneurosome fractions.

FIG. 7, comprising FIGS. 7A through 7B, demonstrates protein translationfrom FLAG-tagged CDC pre-mRNA substrate in isolated dendrites. FIGS. 7A,7D, and 7G depict phase contrast images of whole neurons before cellbody removal. Corresponding phase contrast images of isolated dendritestransfected with; no RNA (FIG. 7B), mature CDC RNA (FIG. 7E), orunspliced, CDC pre-RNA (FIG. 7H), and subsequent immunodetection ofprotein translation with antibody for FLAG and DAB visualization areshown in FIGS. 7C, 7F, and 7I, respectively. Black arrows indicateisolated, transfected dendrites before and after immunodetection of FLAGprotein translation. White arrows point out areas previously occupied bycell bodies before dissection.

FIG. 8, comprising FIGS. 8A through 8J, is a series of imagesdemonstrating that FLAG protein can be translated from alternativelyspliced CDC RNAs. FIGS. 8G, 8H, 8I, and 8J depict schematics of CDCpre-RNA and processed CDC RNAs used in the isolated dendritetransfection assay. CDC RNAs were synthesized from alternatively splicedvariants 1, 6, and 45 (see FIG. 1, also set forth in Table 1) and theseRNAs were transfected into isolated dendrites and subjected toimmunohistochemistry. FLAG immunoreactivity was visualized with DAB.Red, represents premature termination codon produced in first readingframe by alternative splicing. White, represents the initiatormethionine of the second cistron. FIGS. 8A, 8C, and 8E are brightfieldimages of isolated dendrites transfected with 236 bp, 181 bp, and 146 bpRNAs, respectively and subsequent phase contrast images are depictedFIGS. 8B, 8D, and 8F, respectively. Black arrows indicate isolateddendrites and areas of varied FLAG immunoreactivity.

FIG. 9 is a schematic depicting clustering of CDC RNA splice junctions.FIG. 9 shows a linear representation of splice acceptor/donor clustersites relative to the canonical splice pair AG/GU . . . AG.Acceptor/donor pairs are matched by their respective color.

FIG. 10, comprising FIGS. 10A through 10E, depicts control data for GFPfusion constructs. Control experiments we conducted using the OlympusFluoview FV1000 to illustrate the specificity of the GFP fusionconstructs.

FIG. 11, comprising FIGS. 11A and 11B, illustrates isolated dendriteviability evaluation using mitochondrial function measurement.

DETAILED DESCRIPTION OF THE INVENTION

The morphology of a dendrite, and the manner of interaction of adendrite with surrounding structures, including other neurons andneurites, including axons and other dendrites, plays a significant rolein the electrophysiological characteristics of the dendrite. Dendriticinteractions also play a role with respect to the “connectedness” of aneuron within a synaptic network.

In the present invention, the presence of nuclear RNA splicing machineryin dendrites is shown for the first time using multiple localizationprocedures. It is also shown for the first time herein that whenisolated dendrites are transfected with a δ-crystallin (CDC) pre-RNA orluciferase reporter RNA containing an intron, splicing at canonical andcryptic splice acceptor/donor sites is obtained. Additionally, in vitrosynaptoneurosome experiments set forth herein for the first time showthat the cytoplasmic subcellular fraction contains a similar complementof splicing constituents that is able to splice CDC pre-RNA. Analysis ofthe CDC pre-mRNA spliced RNAs reveals that a subset of thedendritically-spliced transcripts can be locally translated.

Therefore, the present application features methods for dendritic-localsplicing of RNA. This is because, as shown herein for the first time,RNA splicing can occur extranuclearly, and in particular, in thecytoplasm of a cell. The invention also features methods of remodelingthe electrochemical and the physical structure of a dendrite usingmethods related to dendrite-local RNA splicing. Further, the inventionfeatures methods of remodeling the electrochemical and the physicalstructure of a dendrite using methods related to dendrite-local RNAsplicing and subsequent dendrite-local protein translation.

The present invention also features methods of remodeling theelectrochemical and the physical structure of a dendrite using methodsrelated to dendrite-local RNA splicing any mammalian cell cytoplasm ornon-nuclear cellular compartment or fraction, as well as methods ofremodeling the electrochemical and the physical structure of a dendriteusing methods related to dendrite-local RNA splicing and subsequentdendrite-local protein translation in any mammalian cell cytoplasm ornon-nuclear cellular compartment or fraction.

It is also a feature of the present invention to provide methods for thecytoplasmic splicing of RNA in dendrites. Further, the inventionprovides methods cytoplasmic splicing and translation of RNA in anymammalian cell cytoplasm or non-nuclear cellular compartment orfraction.

DEFINITIONS

Unless defined otherwise, all technical and scientific terms used hereingenerally have the same meaning as commonly understood by one ofordinary skill in the art to which this invention belongs. Generally,the nomenclature used herein and the laboratory procedures in cellculture, molecular genetics, organic chemistry, and nucleic acidchemistry and hybridization are those well known and commonly employedin the art.

Standard techniques are used for nucleic acid and peptide synthesis. Thetechniques and procedures are generally performed according toconventional methods in the art and various general references (e.g.,Sambrook and Russell, 2001, Molecular Cloning, A Laboratory Approach,Cold Spring Harbor Press, Cold Spring Harbor, N.Y., and Ausubel et al.,2002, Current Protocols in Molecular Biology, John Wiley & Sons, NY),which are provided throughout this document.

The nomenclature used herein and the laboratory procedures used inanalytical chemistry and organic syntheses described below are thosewell known and commonly employed in the art. Standard techniques ormodifications thereof, are used for chemical syntheses and chemicalanalyses.

The articles “a” and “an” are used herein to refer to one or to morethan one (i.e., to at least one) of the grammatical object of thearticle. By way of example, “an element” means one element or more thanone element.

As used herein, amino acids are represented by the full name thereof, bythe three letter code corresponding thereto, or by the one-letter codecorresponding thereto, as indicated in the following table: Full NameThree-Letter Code One-Letter Code Aspartic Acid Asp D Glutamic Acid GluE Lysine Lys K Arginine Arg R Histidine His H Tyrosine Tyr Y CysteineCys C Asparagine Asn N Glutamine Gln Q Serine Ser S Threonine Thr TGlycine Gly G Alanine Ala A Valine Val V Leucine Leu L Isoleucine Ile IMethionine Met M Proline Pro P Phenylalanine Phe F Tryptophan Trp W

As used herein, to “alleviate” a disease, disorder or condition meansreducing the severity of one or more symptoms of the disease, disorderor condition.

An “isolated nucleic acid” refers to a nucleic acid segment or fragmentwhich has been separated from sequences which flank it in a naturallyoccurring state, e.g., a DNA fragment which has been removed from thesequences which are normally adjacent to the fragment, e.g., thesequences adjacent to the fragment in a genome in which it naturallyoccurs. The term also applies to nucleic acids which have beensubstantially purified from other components which naturally accompanythe nucleic acid, e.g., RNA or DNA or proteins, which naturallyaccompany it in the cell. The term therefore includes, for example, arecombinant DNA which is incorporated into a vector, into anautonomously replicating plasmid or virus, or into the genomic DNA of aprokaryote or eukaryote, or which exists as a separate molecule (e.g.,as a cDNA or a genomic or cDNA fragment produced by PCR or restrictionenzyme digestion) independent of other sequences. It also includes arecombinant DNA which is part of a hybrid gene encoding additionalpolypeptide sequence.

In the context of the present invention, the following abbreviations forthe commonly occurring nucleic acid bases are used. “A” refers toadenosine, “C” refers to cytidine, “G” refers to guanosine, “T” refersto thymidine, and “U” refers to uridine.

A “polynucleotide” means a single strand or parallel and anti-parallelstrands of a nucleic acid. Thus, a polynucleotide may be either asingle-stranded or a double-stranded nucleic acid.

The term “nucleic acid” typically refers to large polynucleotides.

The term “oligonucleotide” typically refers to short polynucleotides,generally, no greater than about 50 nucleotides. It will be understoodthat when a nucleotide sequence is represented by a DNA sequence (i.e.,A, T, G, C), this also includes an RNA sequence (i.e., A, U, G, C) inwhich “U” replaces “T.”

Conventional notation is used herein to describe polynucleotidesequences: the left-hand end of a single-stranded polynucleotidesequence is the 5′-end; the left-hand direction of a double-strandedpolynucleotide sequence is referred to as the 5′-direction.

The direction of 5′ to 3′ addition of nucleotides to nascent RNAtranscripts is referred to as the transcription direction. The DNAstrand having the same sequence as an mRNA is referred to as the “codingstrand”; sequences on the DNA strand which are located 5′ to a referencepoint on the DNA are referred to as “upstream sequences”; sequences onthe DNA strand which are 3′ to a reference point on the DNA are referredto as “downstream sequences.”

A “portion” of a polynucleotide means at least at least about twentysequential nucleotide residues of the polynucleotide. It is understoodthat a portion of a polynucleotide may include every nucleotide residueof the polynucleotide.

A “recombinant polypeptide” is one which is produced upon expression ofa recombinant polynucleotide.

“Polypeptide” refers to a polymer composed of amino acid residues,related naturally occurring structural variants, and syntheticnon-naturally occurring analogs thereof linked via peptide bonds,related naturally occurring structural variants, and syntheticnon-naturally occurring analogs thereof. Synthetic polypeptides can besynthesized, for example, using an automated polypeptide synthesizer.

The term “protein” typically refers to large polypeptides.

The term “peptide” typically refers to short polypeptides.

Conventional notation is used herein to portray polypeptide sequences:the left-hand end of a polypeptide sequence is the amino-terminus; theright-hand end of a polypeptide sequence is the carboxyl-terminus.

As used herein, to “treat” means reducing the frequency with whichsymptoms of a disease, disorder, or adverse condition, and the like, areexperienced by a patient.

As the term is used herein, “modulation” of a biological process refersto the alteration of the normal course of the biological process.

As used herein, the term “remodel” relates to an alteration of the stateor condition of something from a previous state or condition. Forexample, a neural network is “remodeled” as a result of a procedure ortreatment if at least one neural connection or interface is changed froma previous state or condition as a result of the procedure or treatment.

The term “dendrite contact,” as used herein, indicates physical contactof a dendrite with another physiological structure, including, but notlimited to a second dendrite, an axon, a neurite, or a soma. The term“dendrite interaction” or “interaction with a dendrite” indicates atleast one of chemical and physical contact of a dendrite with anotherphysiological structure, but does not require physical contact with adendrite.

As used herein, the term “synaptic network” refers to an interconnectednetwork of neurons, and may include other components. The term “neuralnetwork” also refers to a network of neurons, and may include othercomponents.

Description of the Invention

A. Methods of Splicing and Translating RNA

The present invention features a method of splicing RNA in a dendrite.It has been shown for the first time herein that, using a dendrite,which is a specialized extension of a neuron involved in interconnectingother neurons, RNA can be spliced outside of the nucleus. It has alsobeen shown for the first time herein that, using the splicing capabilityof dendrites, RNA can be spliced outside of the nucleus. RNA splicing isuseful for various purposes in the field of genetics and molecularbiology, including the production of stable and properly organizedtranscripts, as well as the production of correctly-spliced andfunctional translated gene products. RNA splicing as described in thepresent invention is also useful for the genetic treatment of diseasesin which the proper transcript is unstable or reactive. Delivery of astable, unspliced transcript to a cell or an organism can enable thesubsequent splicing and translation of the desired transcript.

In one embodiment of the invention, a method of splicing an RNA includestransfecting a dendrite with an RNA comprising at least one intron,wherein the dendrite comprises at least one component of a spliceosome,and the component of a spliceosome is capable of splicing an RNA. Thetransfected RNA is spliced by the spliceosome component within thedendrite. In one aspect of the invention, the dendrite is an isolateddendrite. That is, the dendrite is isolated from the rest of a neuron.

In an aspect of the invention, an RNA splicing donor/acceptor pair is acanonical pair. In another aspect of the invention, an RNA splicingdonor/acceptor pair is an atypical pair. In yet another aspect of theinvention, an RNA splicing donor/acceptor pair is a cryptic pair.

In an embodiment of the invention, an RNA comprising at least one intronis a pre-RNA. In one aspect, the RNA is a pre-mRNA. As discussedelsewhere herein, a pre-RNA is any RNA that can be processed to giverise to a molecularly distinct RNA molecule. Examples of pre-RNAsinclude, but are not limited to, an RNA that can be spliced to producean RNA splice product that is shorter in length. As will be understoodby the skilled artisan, intron-containing RNA useful in the presentinvention can be prepared in any number of ways, and the method ofpreparation of RNA should not be considered limiting. By way of anon-limiting example, RNA useful in the present invention may beprepared by methods including isolation of native RNA from a cell,isolation of RNA from a recombinant system in which a recombinant DNAconstruct was used to transcribe RNA, or from an RNA virus or arecombinant RNA virus (eg., rhinovirus, hepatitis C).

In one embodiment of the invention, RNA comprising at least one intronis derived from a recombinant DNA construct. A construct useful in thepresent invention is designed to provide a transcribed RNA comprising atleast one splice site. Constructs useful in the present inventioninclude, but are not limited to, pEGFP-N1, pDsRed-N1, SF1/mBBP-GFP,U2AF65-GFP, a GFP construct, a DsRed construct, a histone 2B-YFPconstruct. In another embodiment of the invention, a construct can alsobe engineered to comprise at least one intron. In one aspect, aconstruct is engineered so that RNA splicing produces a detectableproduct. By way of a non-limiting example, a construct can be engineeredsuch that splicing results in an output of fluorescence.

In an embodiment of the invention, at least one spliceosome component ispresent in a cytoplasm useful for splicing an RNA according to a methodof the present invention. In one aspect, a spliceosome component isidentified by a detection process, as described in detail in theExperimental Examples of the present application. In another aspect, aspliceosome component can be recombinantly engineered to exist in acytoplasm useful for splicing an RNA according to a method of thepresent invention. Such recombinant techniques are well-known in theart, and will not be discussed in detail herein. For example, theinvention encompasses expression vectors and methods for theintroduction of exogenous DNA into cells with concomitant expression ofthe exogenous DNA in the cells such as those described, for example, inSambrook et al. (2001, Molecular Cloning: A Laboratory Manual, ColdSpring Harbor Laboratory, New York), and in Ausubel et al. (1997,Current Protocols in Molecular Biology, John Wiley & Sons, New York).

By way of a non-limiting example, spliceosome components useful in thepresent invention include, but are not limited to, Y14, Magoh, RNPS1,SC-35, SF2, U2AF65, Smith antigen, pan-SR antigen, U1 snRNP, U2 snRNP,U4 snRNP, U5 snRNP and U6 snRNP.

In another embodiment of the invention, a dendrite is a component of aneuron. That is, the dendrite is attached to a neuron comprising atleast a soma. In one embodiment, the neuron is isolated. In anotherembodiment, the neuron is maintained in culture. In yet another aspectof the invention, a dendrite is a component of a neuron, wherein theneuron is in vivo, in a living organism. In one embodiment, the neuronis a part of a neural network. In another embodiment, the neuron is atransplanted neuron. In one aspect, the neuron is a transplanted neuronthat is not a part of a neural network, but has the potential to bestimulated to integrate into an existing neural network.

This is because it has been shown herein for the first time thatdendrites contain components of the RNA spliceosome and of the cellulartranslation machinery, and that RNA can be spliced and subsequentlytranslated outside of the nucleus. In one embodiment, the presentinvention features a method of splicing an intron-containing RNA in thecytoplasm of a dendrite, followed by the translation of the spliced RNAto the corresponding protein.

In one embodiment, a method of translating a spliced RNA includestransfecting a dendrite with an RNA comprising at least one intron,wherein the dendrite comprises at least one component of a spliceosome,and the component of a spliceosome is capable of splicing an RNA. Thetransfected RNA is spliced by the spliceosome component within thedendrite, and the spliced RNA is translated by the components of thetranaslation machinery located within the dendrite.

In another embodiment of the invention, a method of translating aspliced RNA involves an isolated synaptoneurosome. In an embodiment ofthe invention, a method of translating a spliced RNA includes contactinga synaptoneurosome with an RNA comprising at least one intron, whereinthe synaptoneurosome comprises at least one component of a spliceosome,and the component of a spliceosome is capable of splicing an RNA. Thetransfected RNA is spliced by the spliceosome component within thesynaptoneurosome, and the spliced RNA is translated by the components ofthe tranaslation machinery located within the synaptoneurosome.

In an aspect of the invention, the dendrite in which an RNA istranslated is an isolated dendrite. In another aspect of the invention,a dendrite in which an RNA is translated is a component of a neuron. Inone embodiment, the neuron is isolated. In another embodiment, theneuron is maintained in culture. In yet another aspect of the invention,a dendrite in which an RNA is translated is a component of a neuron,wherein the neuron is in vivo, in a living organism. In one embodiment,the neuron is a part of a neural network. In another embodiment, theneuron is a transplanted neuron. In one aspect, the neuron is atransplanted neuron that is not a part of a neural network, but has thepotential to be stimulated to integrate into an existing neural network.

This is further because, as described in detail elsewhere herein, an RNAcan be spliced and translated extranuclearly, within a dendrite.Translation of a particular spliced transcript locally within a dendritecan be used to remodel the dendrite. That is, the administration of aspecific intron-containing RNA to a dendrite can be used to direct thelocal production of a protein that can be used to effect a change in thestructure, biology, or electrophysiochemistry of a dendrite. By way of anon-limiting example, an intron-containing RNA is transfected into adendrite, whereby the transfected RNA is specifically spliced, and thespliced transcript is translated locally to produce a protein that isuseful for altering the physical structure and arrangement of thedendrite, thereby altering the neural connections of the dendrite. Suchproteins include, but are not limited to, cadherin, BDNF receptor, andneurexin.

Therefore, the present invention also features a method of remodeling adendrite, wherein the method includes transfecting a dendrite with anRNA comprising at least one intron, wherein the dendrite comprises atleast one component of a spliceosome, and the component of a spliceosomeis capable of splicing an RNA. The transfected RNA is spliced by thespliceosome component within the dendrite, and the spliced RNA istranslated by the components of the tranaslation machinery locatedwithin the dendrite. The protein produced by the translationconsequently effects remodeling of the dendrite. In one aspect of theinvention, the dendrite remodeling includes a physical alteration orrestructuring of the dendrite. In another aspect of the invention, thedendrite remodeling includes an alteration of the chemical nature of thedendrite. In yet another aspect of the invention, the dendriteremodeling includes an alteration of the electrophysiochemical nature ofthe dendrite. In still another aspect of the invention, the dendriteremodeling includes an alteration of more than one property of thedendrite, such as, but not limited to, a physical restructuring of thedendrite that subsequently results in an alteration of the biologicalproperties of the dendrite.

RNAs encoding proteins useful for remodeling a dendrite or a synapticnetwork include, but are not limited to, RNAs encoding cadherin,neurexin, synaptophysin, tubulin, microtubule associated proteins, andactin. As will be understood by the skilled artisan, when armed with thepresent application, RNAs encoding any protein known to be involved inthe growth, homeostasis or remodeling of a dendrite are useful in thepresent invention. As will be understood by the skilled artisan, suchRNAs may be pre-RNAs, and may be spliced to form the final useful RNAmolecule.

One of skill in the art will understand, when armed with the presentdisclosure, that a multitude of properties of a dendrite, and byassociation, of a neuron, can be affected by the methods of the presentinvention. While not wishing to be bound by any particular theory, oneof skill in the art will understand that a method of the presentinvention is useful for the growth of a synaptic network, by way ofdendrite stimulation and remodeling, to form new and additional neuronalconnections through dendrite remodeling. As will be understood by thedisclosure set forth herein, such neural network growth and/orremodeling is useful in vitro, in a neuronal cell culture, or in vivo,in a patient in need of neural network growth and/or remodeling.Conditions for which neural network remodeling is useful include, butare not limited to, neurodegenerative diseases, such as Parkinson'sdisease, Alzheimers disease, Huntington's disease, fragile X disease,Downs' syndrome, and neuropsychiatric illnesses such as depression,schizophrenia, and schizo-affective disorders.

Therefore, the present invention also features a method of remodeling asynaptic network, wherein the method includes transfecting a dendritewith an RNA comprising at least one intron, wherein the dendritecomprises at least one component of a spliceosome, and the component ofa spliceosome is capable of splicing an RNA. The transfected RNA isspliced by the spliceosome component within the dendrite, and thespliced RNA is translated by the components of the translation machinerylocated within the dendrite. The protein produced by the translationconsequently effects remodeling of the dendrite, which results inremodeling of a synaptic network. In particular, the synaptic networkcomprising interaction with the transfected dendrite will thereby beremodeled.

As set forth in greater detail elsewhere herein, dendrite remodeling,and therefore, remodeling of the synaptic network comprising interactionwith the transfected dendrite, includes one or more of a physicalalteration or restructuring of the dendrite, alteration of the chemicalnature of the dendrite, alteration of the electrophysiochemical natureof the dendrite, and alteration of the biological properties of thedendrite.

While the present invention is described in relation to theextranuclear, cytoplasmic splicing and translation of an RNA in relationto a dendrite, the skilled artisan reading the present disclosure willunderstand that the present invention is equally applicable to any cell.That is, the skilled artisan will understand that the methods of thepresent invention are equally applicable to use in the cytoplasm of anymammalian cell, including, but not limited to, human, primate, mouse,rat, equine, sheep, goat, pig and dog, among others. Further, theskilled artisan will understand that the methods of the presentinvention are based upon the phenomenon of RNA splicing, and subsequenttranslation of the spliced RNA, which processes are essential componentsof all mammalian cells.

Based on the disclosure set forth herein, the skilled artisan willunderstand how to identify components of the spliceosome extranuclearly,in the cytoplasm, and further, will understand how to identifycomponents of the translation machinery in the cytoplasm. Further still,the skilled artisan, when equipped with the disclosure of the presentinvention, will understand how to assay for extranuclear RNA splicingand for translation of the spliced RNA product. Based on the extensivedisclosure set forth herein, the routineer will understand that thepresent disclosure guides the skilled artisan to assay for extranuclearRNA splicing and for translation of the spliced RNA product usingtechniques available in the art and within the realm of ordinary androutine experimentation.

Therefore, the present invention also includes a method of splicing anRNA, including transfecting a cell with an RNA comprising at least oneintron, wherein the cell comprises at least one component of aspliceosome in the cytoplasm, and the component of a spliceosome iscapable of splicing an RNA. The transfected RNA is spliced by thespliceosome component within the cytoplasm of the cell. In one aspect ofthe invention, the cell is an isolated cell. As will be understood basedon the discussion set forth herein, the cell can be any mammalian cellcomprising a cytoplasm.

Further, in an embodiment of the invention, a method of translating aspliced RNA includes transfecting a cell with an RNA comprising at leastone intron, wherein the cell comprises at least one component of aspliceosome in the cytoplasm, and the component of a spliceosome iscapable of splicing an RNA. The transfected RNA is spliced by thespliceosome component within the cytoplasm, and the spliced RNA istranslated by the cell. In another embodiment, the spliced RNA istranslated by the components of the translation machinery located withinthe cytoplasm. In one aspect of the invention, the cell in which aspliced RNA is translated is an isolated cell. In another aspect of theinvention, a cell in which a spliced RNA is translated is a component ofa cell culture. In yet another aspect, a cell in which a spliced RNA istranslated is part of a living organism, including, but not limited to,a human.

B. Methods of Assaying for the Presence of Spliceosome Components in theCytoplasm of a Cell

The present invention further includes a method of identifying afunctional complement of spliceosome components in a cell. In oneembodiment, a method of identifying a functional complement ofspliceosome components in a cell comprises the introduction of an RNA,comprising at least one intron, into a non-nuclear compartment of a cellor of a non-nuclear sub-cellular fraction. In one aspect, the methodcomprises the step of detection of any splice products resulting fromthe administration of an RNA. In another aspect, the method comprisesthe step of detection of any protein produced as a result of theproduction of splice products resulting from the administration of anRNA.

By way of a non-limiting example, a cryptic splice site in exon 7 of theElaPDH gene binds with high affinity to SC35 of the spliceosomemachinery (Gabut et al., 2005, MCB 25:3286-3294). A construct thatcontains this splice site can be used according to a method of thepresent invention to produce a splice product that can be detected in anon-nuclear compartment of a cell or in a non-nuclear sub-cellularfraction.

EXPERIMENTAL EXAMPLES

The invention is further described in detail by reference to thefollowing experimental examples. These examples are provided forpurposes of illustration only, and are not intended to be limitingunless otherwise specified. Thus, the invention should in no way beconstrued as being limited to the following examples, but rather, shouldbe construed to encompass any and all variations which become evident asa result of the teaching provided herein.

Materials and Methods

Immunohistochemical detection of splicing proteins in neurons: E18 rathippocampal neurons were prepared as previously described. 8-10 dayslater, cultured neurons were washed in pre-warmed Hank's balanced saltsolution supplemented with 850 mg/Liter sodium bicarbonate, 20 mM HEPES,pH 7.4, and 1 mM sodium pyruvate then 1×PBS/0.12 M sucrose for oneminute each. Cells were fixed in 4% paraformaldehyde (ElectronMicroscopy Sciences) in PBS/0.12 M sucrose for 7 minutes and washedthree times in PBS/0.12 M sucrose and two times in PBS/5mM MgCl2 for 5minutes each. Cells were then permeabilized with 0.3% Triton-X 100/PBSfor 5 minutes, washed three times in PBS/5 mM MgCl2 for 5 minutes each,and stored in blocking solution (10% goat serum (Sigma), 0.1% fishgelatin, 0.1% Tween 20, in PBS] for 2 hrs. Cells were incubatedovernight at 4° C. with the following primary antibodies diluted in 1%blocking solution/PBST(0.1% Tween/PBS): anti-Sm proteins (5 μg/ml, LabVision Corporation), anti SF2 (10 μg/ml, Zymed Laboratories INC. (thisantibody does not recognize SC-35 or SF2)), anti-SC-35 (1:500, AccurateChemical and Scientific Corporation), anti-SR (10 ug/ml ZymedLaboratories INC) and anti-U2AF65(10 ug/ml Zymed Laboratories INC).Cells were then washed 3 times in PBST 5 minutes each and incubated for2 hours in goat anti-mouse Qdot 525 (1:50, Quantum Dots, QDot Corp.).Neurons were washed 3 times in PBST for 10 minutes each, thensequentially labeled with rabbit polyclonal anti-MAP2 antibody (1:4000,kindly provided by Craig Garner, Stanford University) and a secondprimary antibody in 1% blocking solution/PBST overnight at 4° C. thenwashed 3 times in PBS/Tween and incubated 2 hours in Cy5 secondaryantibody (1:250) and Qdot 605 (Quantum Dot 1:50). Following incubation,cells were washed 3 times in PBST, mounted with vectashield, andcoverslips sealed with nail polish. Antibody dilution series were donewith each antibody to determine the optimal dilution to use for thesestudies and secondary antibodies, absent primary antibodies showed nospecific binding (data not shown). Confocal imaging was performed usinga Fluoview 1000 confocal microscope (Olympus) and a three-channelconfocal microscope (Prairie Technologies, WI) attached to an OlympusBX50 fixed-stage upright microscope, with excitation at 458 nm and 633nm for imaging quantum dot (525,605) and Alexa 633, respectively. Forselective emission collection, 515±15 nm and 585±20 nm band pass filterswere used for quantum dot imaging. To prevent bleed through, eachquantum dot was imaged to setup laser power and photomultiplier gainbefore imaging triple-labeled samples. To confirm a lack ofbleed-through between channels and confirm specificity of reactivity,samples were imaged in parallel when primary antibody was omitted. Insome experiments we collected the emission spectra and confirmed theidentity of each label. Z-stacks of confocal images were acquired byincrementing each image by 0.2 μm. Images were processed using Metamorphsoftware (Universal Imaging, West Chester, Pa.). Other immunofluorescentimages were taken on a Zeiss Axiovert 200 microscope attached to anOrca-ER camera (Hamamatsu) and processed with Axiovision 3.1 software(Zeiss), an Olympus IX81 microscope using FluoView, or an Olympus IX71microscope using an Olympus DP12 camera.

Generation of spliceosome-related RBP constructs: cDNAs encoding ratMagoh, RNPS1, SF1/mBBP, USAF65, UAP56, and Y14 were isolated from wholerat brain by reverse transcription polymerase chain reaction using PfuTurbo (Stratagene). Primers were designed according to the previouslyreported mouse or human sequences in GenBank. Primers for reIF4A3 are5′-AATGAATTCGCCACCATGGCGGCTAACGCCACGATGGCG-3′ (SEQ ID NO: 1) (sense;underlined nucleotides depict EcoRI site) and5′-ATTTGGATCCCGAATTAGGTCAGCCACATTCATGGG-3′ (SEQ ID NO:2) (antisense;underlined nucleotides depict BamHI site). Primers for rMagoh are5′-AATAAGCTTGCCACCATGGAGAGTGACTTTTACCTGCGT -3′ (SEQ ID NO:3) (sense;underlined nucleotides depict HinDIII site) and5′-ATTGACCGGTGGGATTGGTTTAATCTTGAAGTGTAA-3′ (SEQ ID NO:4) (antisense;underlined nucleotides depict AgeI site). Primers for rRNPSI are5′-AATAAGCTTGCCACCATGGATTTATCAGGAGTGAAAAAG-3′ (SEQ ID NO:5) (sense;underlined nucleotidesdepict HindDIII site) and5′-ATTGACCGGTGGGAGCAGCCGTGAACCAACAGT-3′ (SEQ ID NO:6) (antisense;underlined nucleotides depict AgeI site). Primers for rSF1/mBBP are5′-AATGCTAGCGCCACCATGGCGACCGGAGCGAACGCCACG-3′ (SEQ ID NO:7) (sense;underlined nucleotides depict NheI site) and5′-ATTTGGATCCCAATGGGCGCGGAAAGTCCTCAC-3′ (SEQ ID NO:8) (antisense;underlined nucleotides depict BamHI site). Primers for rU2AF65 are5′-AATAAGCTTGCCACCATGGACTTCTTCAACGCCCAGATG-3′ (SEQ ID NO:9) (sense;underlined nucleotides depict HinDIII site) and5′-ATTTGGATCCCAGAAGTCCCGACGGTGGTACGA-3′ (SEQ ID NO: 10) (antisense;underlined nucleotides depict BamHI site). Primers for rUAP56 are5′-AATAAGCTTGCCACCATGGCAGAGAACGATGTGGACAAT-3′ (SEQ ID NO:11) (sense;underlined nucleotides depict HinDIII site) and5′-ATTTGGATCCCGTGTCTGTTCAATGTAGGAGGA (SEQ ID NO:12) (antisense;underlined nucleotides depict BamHI site). Primers for rY14 are5′-AATAAGCTTGCCACCATGGCGGACGTGCTGGATCTTCAC-3′ (SEQ ID NO: 13) (sense;underlined nucleotides depict HinDIII site) and5′-ATTTGGATCCCGACGGCGTCTCCGGTCTGGACTCCT-3′ (SEQ ID NO:14) (antisense;underlined nucleotides depict BamHI site). The PCR products weredigested with the appropriate restriction enzyme and inserted intopEGFP-N1 or pDsRed-N1 (Clontech). All sequences were verified bysequencing and contain the full coding region in-frame with either GFPor DsRed.

DNA/Ca2+-phosphate coprecipitation transfection of DNA constructs intoprimary neuronal cell cultures: Primary hippocampal neurons werecultured for 7-10 days in vitro and then transfected as described byPark et al. with minor modifications (Park et al., 2004, Neurosci. Lett.361:220-4 [36]).

In situ hybridization of U1 RNA: Hippocampal neurons were fixed in 4%paraformamide (10 min), washed 3×5 min in 0.01% triton-X100 PBS (PBST),washed 7 min in 0.2% triton-x100, PBS, and washed 2×5 min in PBS. After3 hours in prehybridization buffer, at 50° C., hybridization buffer (50%formamide, 5×SSC, 1× Denhart's, 8% Dextran Sulfate, 10 mM DTT, yeasttRNA (50 μg/ml)) with 5 ng probe/μl and 8% dextran sulfate was added at50° C. overnight. These hybridization conditions are quite stringent.Cells were washed 2×5 min 2×SSC at 50° C., 2×10 min 0.22×SSC at RT, and1×30 min PBS1 μg/ml RNase A. Cells were then washed 2×5 min in PBS,blocked in PBST+10% goat serum for 30 min. Digoxigenin-labeled riboprobeantisense to bases 20 to 70 of U1 rat snRNA or GAD65 mRNA were detectedwith alkaline-phosphatase conjugated anti-DIG antibodies and visualizedwith BCIP/NBT. Antisense and RNase A controls were also performed.

CDC pre-RNA preparation and RNA transfection of neurons and dendrites:Pre- or mature CDCRNAs (pcDNA3 plasmid construct containing insert ofexons 14 and 15 of the CDC gene either with or lacking intermittentintron sequence and flanked with FLAG epitope; these constructs werekindly provided by Gideon Dreyfuss) were transcribed using Ambion's T7mMessage mMachine kit. Approximately 1 μg of RNA was mixed with 5 μl ofGeneporter (Gene Therapy Systems) and incubated for 10 minutes at roomtemperature then stored on ice until use. Isolated dendrites wereprepared by mechanical severing of the process from the cell soma andRNA/lipid mixtures applied to dendrites as previously described.Following transfection, (RS)-3,5-dihydroxy-phenylglycine (DHPG, Tocris)was added to a final concentration of 20 μM in order to stimulatetranslation and isolated dendrites were incubated at 37° C. for onehour. Dendrites were washed four times in physiological buffer and fixedin 4% paraformaldehyde/PBS for 20 minutes then washed in PBS.

PCR Analysis of dendritic CDC RNA splice products: After transfection ofhippocampal neurons with CDC RNA, dendrites were picked individually anda reverse transcription reaction performed using primer ‘sp6’. Aninitial PCR reaction was performed using primers sp6 and 5A, thenanalyzed by gel electrophoresis. Successful amplification of mRNAresults in the presence of a major 476 bp fragment, with splicedproducts. The area immediately below the 476 bp band is excised and gelpurified, then reamplified utilizing the 5A primer and nested primerFLAG, then analyzed by gel electrophoresis. Samples displaying apositive splicing activity produce DNA fragments ranging in size from125 bp to 476 bp. These fragments were gel purified, cloned andsequenced. Reverse Transcription reaction: AMV-RT (Cape Cod) 42° C. 30min. DNA amplifications were performed with Accuprime PFX DNA polymerase(Invitrogen): 1×95° C. 2 min, 40×95° C. 15 sec, 47° C. 30 sec, 68° C. 30sec, 1×68° C. 7:00 min. Gel extractions were performed with the Qiaquickgel extraction kit (Qiagen). Fragments were A-tailed with Amplitaq(Perkin-Elmer) 10 min 68° C., then ligated to pGEM-T-Easy (Promega).Primers: sp6-ATTTAGGTGACACTATAGA (SEQ ID NO: 15),FLAG-TTTATCGTCATCGTCTTTG (SEQ ID NO: 16), 5A-CCAATCGATATACTTAGCC (SEQ IDNO: 17), 5B-GCCAGTGCCAAGCTTGCTGAC (SEQ ID NO: 18).

Synaptoneurosome preparation: Protocol is adapted from (Weiler et al.,1993, Proc. Natl. Acad. Sci. U.S.A. 90:7168-71; Rao et al., 1993, J.Neurochem. 61:835-44 [49, 50]). Briefly, 6-8 week old maleSprague-Dawley rats were sacrificed by cervical dislocation followed byrapid decapitation. After removal of the cerebellum, the brain ishomogenized with a large dounce homogenizer (Wheaton) in cold isolationmedium containing: 320 mM Sucrose, 10 mM Tris-HCl, 1 mM EDTA. Thehomogenate is spun at 3,500 rpm for three minutes and the supernatant iscolleted and re-spun at 10,000 rpm for ten minutes. After re-suspensionof the resultant pellet in 2.5 mls cold isolation medium, the solutionis further homogenized and mixed with 12% Ficoll (Sigma). 7% Ficoll andisolation medium are then slowly layered on top of the homogenate, andsolutions are spun at 27 K RPM for 35 minutes (Beckman L8-55M).Synaptoneurosome fractions are then collected between the 12% and 7%Ficoll layers and kept on ice. For Western blotting, synaptoneurosomesor whole brain tissue is immediately pelleted and lysed in the presenceof protease inhibitors. Equal amounts of protein (as determined byBradfordAssay) are run on NuPAGE 10% Bis-Tris precut gels (Invitrogen),transferred to PVDF membrane BioRad), stained using the antibodieslisted above and visualized using chemiluminescence (PerkinElmer).Beta-tubulin is used as a loading control.

Experimental Example 1 Identification and Characterization of SplicesomeComponents in Neuronal Dendrites.

The spliceosome, which catalyses the ATP dependent removal of intronsfrom nuclear pre-RNA, is a multi-megadalton complex of proteins andsmall nuclear RNAs (snRNA) (Staley et al., 1998, Cell 92:315-26; Juricaet al., 2003, Mol. Cell 12:5-14). Even in the nucleus, the distributionof pre-RNA splicing factors is not uniform. Rather, with discrete sitesof concentration and lower levels of factors diffusely dispersedthroughout the nucleoplasm, speckles (splicing factor compartments) canbe readily identified with an antibody against the spliceosome assemblyfactor SC-35 (Lamond et al., 2003, Nat. Rev. Mol. Cell Biol. 4:605-12).Initially, confocal microscopy was used to determine theimmunofluorescent localization of the non-nuclear splicing factordomains (FIGS. 2A, 2D, and 2G) in primary neuronal dendrites. Expressionoutside of the nucleus clearly was observed as a series of puncta in theperinuclear space and dendrites (Buchhalter et al., 1991, Brain Res.Bull. 26:333-8). It was subsequently determined that other splicingfactors associated with the initiation and commitment steps of pre-RNAsplicing were also detectable in the dendritic arbor. Because SC-35nuclear sites show relatively limited amounts of uridine incorporation,speckles are believed to be storage sites for numerous splicing factorsand serine/arginine (SR)-rich proteins (Moen et al., 1995, Hum. Mol.Genet. 4:1779-89). Using antisera directed against four core componentsof the pre-spliceosome (i.e. SF2, U2AF65, Smith antigen (Sm), and pan-SRantigens), it was determined that these proteins co-exist withdendritically localized SC-35. (Buchhalter et al., 1991, Brain Res.Bull. 26:333-8) Each of these proteins has previously been detected inspeckles of somatic nuclei as well as other nuclear subdomains.

Intrinsic to the pre-spliceosome complex are the Sm proteins thatcombine with snRNAs to form the core constituents of the small nuclearribonucleoprotein particles (snRNPs); U1, U2, and U4-U6 (Will et al.,2001, Curr. Opin. Cell Biol. 13:290-301). In turn, the U1 snRNP isrecruited to the nascent pre-RNA via the interaction between SF2 andintronic RNA sequence. During several other transitions, U2AF65,SF1/mammalian branch point binding protein (SF1/mBBP), and SR-richproteins are subsequently recruited to the branch point sequenceembedded in pre-RNA introns wherein rearrangements lock the U2 snRNPonto the pre-mRNA thereby committing to nuclear pre-RNA splicing (Guthet al., 1999, Mol. Cell Biol. 19:8263-71). When localization of thesesplicing factors was examined, high expression of USAF65 was observed(FIG. 2B), Sm antigen (FIG. 2E), SF2 (FIGS. 2H and 2K), and SR proteins(FIG. 2D) in the nuclei of both neurons and glia. These data areconsistent with previous experiments noting their nuclear distribution.However, in each experiment, small puncta, or granule-like structures,were observed, located in proximal and distal portions of dendrites andtheir branch points. These granule-like bodies (Mattaj et al., 1985,Cell 40:111-8) were also observed in the perinuclear space of bothneurons and glia often juxtaposed with the nuclear envelope (data notshown). For some proteins such as SF2, more granule-like structures wereobserved in the perinuclear space with reduced levels of SF2 punctalocalized in proximal and distal dendrites (Tacke et al., 1995, Embo. J.14:3540-51). The merged confocal images shown in FIGS. 2C, 2F, 2I and 2Lillustrate the co-localization of each of the antigens with speckles orwith each other. The presence of the pre-spliceosome-related antigens inthe dendritic domain of the neurons was confirmed by co-localization ofmicrotubule-associated protein 2 (MAP2) immunofluorescence (inset FIG.2A). MAP2 is a protein marker of the somatodendritic domain of neurons.A predominantly nuclear localization, in contrast, was observed whenusing an anti-histone 3 antibody (see FIGS. 10E and 10F).

Since the traditional pre-RNA splicing complex also employs an RNAcomponent, in situ hybridization (ISH) was performed for U1 RNA on rathippocampal cultures. U1 RNA is the RNA component of the U1 snRNP and iscritical for initiating traditional RNA splicing. ISH revealed thepresence of U1RNA in the nucleus and more disperse localization in thecytoplasm and dendrites of many neurons (FIG. 3A). U1 RNA wasinterspersed along the length of dendrites, with noticeable stainingoccurring greater than 30 μm from the nucleus. Areas of moderate U1staining also appeared at dendritic branch points. Antisense competitioncontrols, where an excess of U1 antisense RNA is added to theprehybridization solution, washed away and labeled U1 antisense RNAannealed, showed only slight staining within the nucleus, potentiallydue to hybridization within multiple copies of the U1 RNA genes in thegenome (FIG. 3B). Additionally, sense controls showed little staining.Dendrites are identified by MAP2 immunostaining (FIGS. 3C and 3D). Theappearance of high levels of U1 RNA staining in the nucleus was expected(Huang et al., 1992, Proc. Natl. Acad. Sci. U.S.A. 89:305-8). By way ofcomparison the presence of U1 RNA in dendrites is contrasted with thedominant somatic localization of GAD65 mRNA in primary hippocampalneurons (FIGS. 3E and 3F). These data showing U1 RNA expression coupledwith the evidence for pre-spliceosome constituents dispersed throughoutthe dendritic cytoplasm indicate that the assembly of functional coresplicing components and more complex splicing mRNPs which would berequired for the processing of select, dendritically localized mRNAs.

Following the initial commitment to splicing, constitutive pre-mRNAprocessing is also accompanied by a series of dynamic changes in theprotein composition of the spliceosome. Three individual subcomplexes ofthe spliceosome (i.e. A (previously referred to as the pre-spliceosome),B*, and C) have recently been purified and their components identifiedby mass spec analysis (Hartmuth et al., 2002, Proc. Natl. Acad. Sci.U.S.A. 99:16719-24; Jurica et al., 2002, Rna 8:426-39; Makarov et al.,2002, Science, 298:2205-8). Having already explored the distribution ofpre-spliceosome components, subcomplexes B* and C were examined.Subcomplex B* temporally represents the mRNP remodeling just prior tothe first transesterification reaction while the catalytic C subcomplexrepresents the splice-intermediate stage following this first chemicalstep. In particular, spliced mRNAs acquire a set of specific proteincomplexes that assemble near exon-exon junctions. Components of thisexon junction complex (EJC), a majority of which are found in subcomplexC (Makarov et al., 2002, Science, 298:2205-8), include SRm160, RNPS1,UAP56, Aly/REF, Upf3, eIF4A3, Y14, and Magoh (Kataoka et al., 2000, Mol.Cell 6:673-82[29]). Assembling 20-24 nucleotides upstream of splicedjunctions, these proteins function in all manner of RNA metabolismincluding mRNA export (Le Hir et al., 2000, Genes Dev. 14:1098-108),coactivation of splicing (Blencowe et al., 1998, Genes Dev. 12:996-1009;Mayeda et al., 1999, Embo. J. 18:4560-70; Mayeda et al. 1999, Mol. CellBiol. 19:1853-63), and nonsense-mediated mRNA decay (NMD) (Kim et al.,2001, Science 293:1832-6). Well-characterized, commercially-availableantibodies, were used to determine the dendritic localization of each ofthe splicing factors examined herein. However, it was not alwaysapparent that the antibodies were able to detect the native proteinswith sufficient sensitivity using immunofluorescence. In part, this islikely due to the large macromolecular mRNP complexes to which they areintrinsic that may sterically hinder antibody access. To circumventthese difficulties and as a secondary confirmation of splicing enzymelocalization, splicing RBPs were engineered, fused in-frame with GFP orDsRed at the carboxy terminus so that the fluorescent marker would betargeted to the subcellular sites where the splicing factors werelocalized. The rat splicing factors were PCR cloned from rat brain cDNAand their identities sequence verified. These splicing enzyme sequenceshave been submitted to Genbank. For complex A-related factors,SF1/mBBP-GFP were generated (FIGS. 4A and 4B), as were U2AF65-GFP fusioncontructs (FIGS. 4C and 4D). The U2AF65-GFP construct was used as asecondary confirmation of immunofluroescence data of splicing factorlocalization performed in FIG. I (see also Table 1). For complex B*, acommercially available antibody to the polypyrmidine tract-bindingprotein associated splicing factor was used (PSF, FIGS. 4E-1 and 4F-1)(Kanai et al., 2004, Neuron 43:513-25). Finally, for complex C, anantibody to Aly/REF (FIGS. 4G-1 and 4H-1) and GFP or DsRed fusionconstructs containing the open reading frame of Magoh (FIGS. 4I and 4J),UAP56 (FIGS. 4L and 4M), Y14 (FIGS. 4N and 4O), and RNPS1 were used. GFPor DsRed fusion constructs were transfected into primary hippocampalcultures at 10-12 days in vitro using a calcium phosphate protocol (Parket al., 2004, Neurosci. Lett. 361:220-4). For low-resolution, whole cellimages, the intensity of the laser was increased to clearly showdendritic signal. In doing so, signal in nuclei is often saturatedwherein specific nuclear subcompartment fluorescence is saturated.Photomontages of Z-stack images illustrate the images obtained duringthese experiments. SF1-mBBP-GFP and U2AF65-GFP showed consistent nuclearlocalization complemented by larger puncta as well as more dispersedgranule-like structures throughout the perinuclear and dendriticcytoplasm in higher magnification images. Of note is the similarity inexpression patterns obtained for U2AF65-GFP and the previous U2AF65immunofluorescence obtained in FIG. 2. Complex B* component PSF showedan altogether separate pattern of localization. As previously described,speckled nuclei were distinctly visible. However, a low but significantcytoplasmic staining was visible in the perinuclear space (see arrows inFIG. 4F-1). Experiments with complex C cofactors showed some variationin expression depending upon the protein. Aly/REF immunofluorescence wasrestricted mostly to nuclei. However, a consistently low level ofdiffuse signal was visible interspersed throughout the dendritic arbor.In comparison, Magoh-GFP, Y14-GFP, UAP56-GFP, and RNPS1-GFP werescattered in granule-like structures in the dendroplasm and speckle-likedomains in the nucleoplasm (see FIG. 10). Experiments performed withanti-Magoh or-Y14 antibodies were used to confirm the pattern of signalobtained with the Magoh- or Y14-GFP constructs. Magoh and Y14 are knownto directly interact and have been shown to be important for oskar mRNAlocalization in Drosophila during oogensis. When co-expressed in primaryneurons, Magoh-GFP and Y14-DsRed show nearly complete co-localization(FIG. 4K). TABLE 1 Sequences of dendritically-spliced andsynaptoneurosome-spliced RNAs. EXON DONOR 5′ INTRON 3′ INTRON EXONACCEPTOR OVERLAP/NOTES CDC-preRNA Transfection of Isolated Dendrites 1TTAAGTGTTGTACAG ATACGAGAGCTCTAG CTTTGAGACACTAAC GTGTCCCCAGGGAGC 0/D (SEQID NO:19) (SEQ ID NO:20) (SEQ ID NO:21) (SEQ ID NO:22) 2 TCTCATTATTTAATGTGGTTGGAGGACACA CATGGAGAAGGCTCT GACCCCTGAGTTGCT 0/C (SEQ ID NO:23) (SEQID NO:24) (SEQ ID NO:25) (SEQ ID NO:26) 3 CTGTGCTCCAGGTTGCCACTGGAGTGATTT AGGAGAACATGGAGA AGGCTCTGACCCCTG 0 (SEQ ID NO:27) (SEQ IDNO:28) (SEQ ID NO:29) (SEQ ID NO:30) 4 TGCCACTGGAGTGAT TTCTACCCTCCAGGTCATGGAGAAGGCTCT GACCCCTGAGTTGCT 0 (SEQ ID NO:31) (SEQ ID NO:32) (SEQ IDNO:33) (SEQ ID NO:34) 5 AAGGTTTCAGCTTCT CATTATTTAATGTGG TCCTTTTGCAGGTCAACAAGGAGAACATGG 0 (SEQ ID NO:35) (SEQ ID NO:36) (SEQ ID NO:37) (SEQ IDNO:38) 6 CGAGAGCTCTAGTCT GGTCCTAACATGAAG TGCAGGTCAACAAGG AGAACATGGAGAAGG0/D (SEQ ID NO:39) (SEQ ID NO:40) (SEQ ID NO:41) (SEQ ID NO:42) 7CATTATTTAATGTGG TTGGAGGACACATTT CTGCCTCTCCTCTTT GCAGGTCAACAAGGA 0 (SEQID NO:43) (SEQ ID NO:44) (SEQ ID NO:45) (SEQ ID NO:46) 8 TTGCGTGTTGTACAGATACGAGAGCTCTAG CTTTGAGACACTAAC GTGTCCCCAGGGAGC 0 (SEQ ID NO:47) (SEQ IDNO:48) (SEQ ID NO:49) (SEQ ID NO:50) 9 CTGGAGTGATTTCTA CCCTCCAGGTAAGGTCTCTCTCTCTGCCTC TCCTCTTTGCAGGTC 0 (SEQ ID NO:51) (SEQ ID NO:52) (SEQ IDNO:53) (SEQ ID NO:54) 10 TTTCTACCCTCCAGG TAAGGTTTCAGCTTC CTCTTTGCAGGTCAACAAGGAGAACATGGA 0 (SEQ ID NO:55) (SEQ ID NO:56) (SEQ ID NO:57) (SEQ IDNO:58) 11 TCTGGTCCTAACATG CTGACCCCTCACTCC AACATGGAGAAGGCTCTGACCCCTGAGTTG 0 (SEQ ID NO:59) (SEQ ID NO:60) (SEQ ID NO:61) (SEQ IDNO:62) 12 CCACTGGAGTGATTT CTACCCTCCAGGTAA CTTTGCAGGTCAACAAGGAGAACATGGAGA 0 (SEQ ID NO:63) (SEQ ID NO:64) (SEQ ID NO:65) (SEQ IDNO:66) 13 ACCTGCTGACTGCTG TGCTCCAGGTTGCCA CAAGGAGAACATGGAGAAGGCTCTGACCCC 0 (SEQ ID NO:67) (SEQ ID NO:68) (SEQ ID NO:69) (SEQ IDNO:70) 14 ATGTGGTTGGAGGAC CATTTTAAGTGTTGT CTCCTCTTTGCAGGTCAACAACGAGAACAT 0 (SEQ ID NO:71) (SEQ ID NO:72) (SEQ ID NO:73) (SEQ ID140:74) 15 AGGTTGCCACTGGAG TGATTTCTACCCTCC GCCTCTCCTCTTTGCAGGTCAACAAGGAGA 0 (SEQ ID NO:75) (SEQ ID NO:76) (SEQ ID NO:77) (SEQ IDNO:78) 16 GTTTCAGCTTCTCAT TATTTAATGTGGTTG ATGGAGAAGGCTCTGACCCCTGAGTTGCTG 0 (SEQ ID NO:79) (SEQ ID N0:80) (SEQ ID NO:81) (SEQ IDNO:82) 17 CTCTAGTCTGGTCCT AACATGAAGACTTGC AACAAGGAGAACATGGAGAAGGCTCTGACC 0 (SEQ ID NO:83) (SEQ ID NO:84) (SEQ ID NO:85) (SEQ IDNO:86) 18 CTGTGCTCCAGGTTG CCACTGGAGTGATTT CAAGGAGAACATGGAGAAGGCTCTGACCCC 0 (SEQ ID NO:87) (SEQ ID NO:88) (SEQ ID NO:89) (SEQ IDNO:90) 19 CTCATTATTTAATGT GGTTGGAGGACACAT AGAAGGCTCTGACCCCTGAGTTGCTGTCTA 0 (SEQ ID NO:91) (SEQ ID NO:92) (SEQ ID NO:93) (SEQ IDNO:94) 20 CTTCTCATTATTTAA TGTGGTTGGAGGACA GGAGAAGGCTCTGACCCCTGAGTTGCTGTC 0 (SEQ ID NO:95) (SEQ ID NO:96) (SEQ ID NO:97) (SEQ IDNO:98) 21 AGTAAGGTTTCAGCT TCTCATTATTTAATGT GTTGTGTCTACTGATCGGGGTAGACTACAA 1 (SEQ ID NO:99) (SEQ ID NO:100) (SEQ ID NO:101) (SEQ IDNO:102) 22 GACTGCTGTGCTCCA GGTTGCCACTGGAGT CTCTTTGCAGGTCAACAAGGAGAACATGGA 1 (SEQ ID NO:103) (SEQ ID NO:104) (SEQ ID NO:105) (SEQID NO:106) 23 TGGAGTGATTTCTAC CCTCCAGGTAAGGTT GCAAATGATAACCTCTCTCTCTGCCTCTCC 1 (SEQ ID NO:107) (SEQ ID NO:108) (SEQ ID NO:109) (SEQID NO:110) 24 TAGTCTGGTCCTAAC ATGAAGACTTGCTCA GAGAAGGCTCTGACCCCTGAGTTGCTGTCT 1 (SEQ ID NO:111) (SEQ ID NO:112) (SEQ ID NO:113) (SEQID NO:114) 25 CATGAAGACTTGCTC ACTCCTACTGCTTGT CGAGAAGGCTCTGACCCCTGAGTTGCTGTC 1 (SEQ ID NO:115) (SEQ ID NO:116) (SEQ ID NO:117) (SEQID NO:118) 26 GGACACATTTTAAGT GTTGTACAGATACCA TCTCTCTCTGCCTCTCCTCTTTGCAGGTCA 1 (SEQ ID NO:119) (SEQ ID NO:120) (SEQ ID NO:121) (SEQID NO:122) 27 GAGTGATTTCTACCC TCCAGGTAAGGTTTC GGAGAAGGCTCTGACCCCTGAGTTGCTGTC 1 (SEQ ID NO:123) (SEQ ID NO:124) (SEQ ID NO:125) (SEQID NO:126) 28 AGGACACATTTTAAG TGTTGTACAGATACG AGGCTCTGACCCCTGAGTTGCTGTCTACTG 1 (SEQ ID NO:127) (SEQ ID NO:128) (SEQ ID NO:129) (SEQID NO:130) 29 TGCTGTGCTCCAGGT TGCCACTGGAGTGAT GACCCCTGAGTTGCTGTCTACTGATCGGGT 1 (SEQ ID NO:131) (SEQ ID NO:132) (SEQ ID NO:133) (SEQID NO:134) 30 GAGTGATTTCTACCC CCCTGTAAGGTTTCA AGAAGGCTCTGACCCCCCTGAGTTGCTGTC 1 (SEQ ID NO:135) (SEQ ID NO:136) (SEQ ID NO:137) (SEQID NO:138) 31 GTTGTACAGATACGA GCTCTAGTCTGGTCC AGGAGAACATGGAGAAGGCTCTGACCCCTG 2 (SEQ ID NO:139) (SEQ ID NO:140) (SEQ ID NO:141) (SEQID NO:142) 32 AGTAAGGTTTCAGCT TCTCATTATTTAATG CAAATGATAACCTCTCTCTCTGCCTCTCCT 2 (SEQ ID NO:143) (SEQ ID NO:144) (SEQ ID NO:145) (SEQID NO:146) 33 AGGTAAGGTTTCAGC CTCTCCTCTTTGCAG AACCTCTCTCTCTGCCTCTCCTCTTTGCAG 2 (SEQ ID NO:147) (SEQ ID NO:148) (SEQ ID NO:149) (SEQID NO:150) 34 TGACTGCTGTGCTCC CAGGTTGCCACTGGA GAGAAGGCTCTGACCCCTGAGTTGCTGTCT 2 (SEQ ID NO:151) (SEQ ID NO:152) (SEQ ID NO:153) (SEQID NO:154) 35 TTTCTACCCTCCAGG TAAGGTTTCAGCTTC AGAACATGGAGAAGGCTCTGACCCCTGAGT 3/A (SEQ ID NO:155) (SEQ ID NO:156) (SEQ ID NO:157) (SEQID NO:158) 36 TGCTGTGCTCCAGGT GCCACTGGAGTGATT GTCTACTGATCGGGTAGACTACAAAGACGA 3 (SEQ ID NO:159) (SEQ ID NO:160) (SEQ ID NO:161) (SEQID NO:162) 37 GATTTCTACCCCCCA TGTAAGGTTTCAGCT CCTTGATGAAGTCCATTCTTTGAGACACTA 3 (SEQ ID NO:163) (SEQ ID NO:164) (SEQ ID NO:165) (SEQID NO:166) 38 GGTTTCAGCTTCTCA TTATTTAATGTGGTT CCTCTTTGCAGGTCAACAAGGAGAACATGG 3 (SEQ ID NO:167) (SEQ ID NO:168) (SEQ ID NO:169) (SEQID NO:170) 39 CTCTAGTCTGGTCCT AACATGAAGACTTGC AAGGCTCTGACCCCTGAGTTGCTGTCTACT 3 (SEQ ID NO:171) (SEQ ID NO:172) (SEQ ID NO:173) (SEQID NO:174) 40 GCTGACTGCTGTGCT CCAGGTTGCCACTGG GACCCCTGAGTTGCTGTCTACTGATCGGGT 4 (SEQ ID NO:175) (SEQ ID NO:176) (SEQ ID N0:177) (SEQID NO:178) 41 GTGATTTCTACCCTC CAGGTAAGGTTTCAG CTCTCTCTCTGCCTCTCCTCTTTGCAGGTC 4 (SEQ ID NO:179) (SEQ ID NO:180) (SEQ ID NO:181) (SEQID NO:182) 42 TGATTTCTACCCTCC TGTAAGGTTTCAGCT TCTCTCTGCCTCTCCTCTTTGCAGGTCAAC 4 (SEQ ID NO:183) (SEQ ID NO:184) (SEQ ID NO:185) (SEQID NO:186) 43 TATTTAATGTGGTTG GAGGACACATTTTAA CTGACCCCTGAGTTGCTGTCTACTGATCGG 4 (SEQ ID NO:187) (SEQ ID NO:188) (SEQ ID NO:189) (SEQID NO:190) 44 GTGATTTCTACCCTC CAGGTAAGGTTTCAG TCTCTGCCTCTCCTCTTTGCAGGTCAACAA 4/A (SEQ ID NO:191) (SEQ ID NO:192) (SEQ ID NO:193) (SEQID NO:194) 45 CTGGTCCTAACATGA AGACTTGCTCACTCC GGGAGCAGCAAATGATAACCTCTCTCTCTG 4/D (SEQ ID NO:195) (SEQ ID NO:196) (SEQ ID NO:197) (SEQID 14O:198) 46 ACCCTCCAGGTAAGG TTTCAGCTTCTCATT TGCAGGTCAACAAGGAGAACATGGAGAAGG 4/A (SEQ ID NO:199) (SEQ ID NO:200) (SEQ ID NO:201) (SEQID NO:202) 47 TTTAATGTGAAGACT TGCTCACTCCTACTG CTGATCGGGTAGACTACAAAGACGATGACC 5/D (SEQ ID NO:203) (SEQ ID NO:204) (SEQ ID NO:205) (SEQID NO:206) 48 TGCTGTGCTCCAGGT TGCCACTGGAGTGAT CTCCTCTTTGCAGGTCAACAAGGAGAACAT 5/A (SEQ ID NO:207) (SEQ ID NO:208) (SEQ ID NO:209) (SEQID NO:210) 49 TCTGGTCCTAACATG AAGACTTGCGTGTTG AACAAGGAGAACATGGAGAAGGCTCTGACC 6/B (SEQ ID NO:211) (SEQ ID NO:212) (SEQ ID NO:213) (SEQID NO:214) 50 GTGCCAAGCTTGCTG ACTGCTGTGCTCCAG ACCCCTGAGTTGCTGTCTACTGATCGGGTA 6 (SEQ ID NO:215) (SEQ ID NO:216) (SEQ ID NO:217) (SEQID NO:218) 51 CTTGCTGACTGCTGT GCTCCAGGTTGCCAC CCCCTGAGTTGCTGTCTACTGATCGGGTAG 6 (SEQ ID NO:219) (SEQ ID NO:220) (SEQ ID NO:221) (SEQID NO:222) 52 TTGCTACTCCTACTG CTTGTTATGACCCCA AGTTGCTGTCTACTGATCGGGTAGACTACA 6 (SEQ ID NO:223) (SEQ ID NO:224) (SEQ ID NO:225) (SEQID NO:226) 53 TCTGGTCCTAACATG AAGACTTGCTCACTC AACAAGGAGAACATGGAGAAGGCTCTGACC 6/B (SEQ ID NO:227) (SEQ ID NO:228) (SEQ ID NO:229) (SEQID NO:230) 54 TTTAATGTGAAGACT TGCTCACTCCTACTG CTGATCGGGTAGACTACAAAGACGATGACG (SEQ ID NO:231) (SEQ ID NO:232) (SEQ ID NO:233) (SEQ IDNO:234) Luciferase-SV40 pre-RNA 1 AAAGTCCAAATTGTA CCAAATTGTAAAATGTACTGTTTTTTCTTA CTCCACACAGGCATA 2 (SEQ ID NO:235) (SEQ ID NO:236) (SEQID NO:237) (SEQ ID NO:238) 2 AAGTCCAAATTGTAA AATGTAACTGTATTCCAGTTATAATCATAA CATACTGTTTTTTCT 2 (SEQ ID NO:239) (SEQ ID NO:240) (SEQID NO:241) (SEQ ID NO:242) 3 TTACGTCGCCAGTCA AGTAACAACCGCGAATAACAGTTATAATCA TAACATACTGTTTTT 3 (SEQ ID NO:243) (SEQ ID NO:244) (SEQID NO:245) (SEQ ID NO:246) 4 CTTACCGGAAAACTC GACGCAAGAAAAATCTGTTTTTTCTTACTC CACACAGGCATAGAG 4 (SEQ ID NO:247) (SEQ ID NO:248) (SEQID NO:249) (SEQ ID NO:250) 5 GATGACGGAAAAAGA GATCGTGGATTACGTCTCCTCCAAAAAAGA AGAGAAAGGTAGAAG 7 (SEQ ID NO:251) (SEQ ID NO:252) (SEQID NO:253) (SEQ ID NO:254) Sequences derived from SynaptoneurosomeSplicing of CDC pre-RNA 1 CTTGCTCACTCCTAC TGCTTGTTATGACCCCTACTGATCGGGTAG ACTACAAAGACGATG 0 (SEQ ID NO:255) (SEQ ID NO:256) (SEQID NO:257) (SEQ ID NO:258) 2 TTTTAAGTGTTGTAC AGATACGAGAGCTCTTCTCTGCCTCTCCTC GATAACCTCTCTCTC 0 (SEQ ID NO:259) (SEQ ID NO:260) (SEQID NO:261) (SEQ ID NO:262) 3 TTCAGCTTCTCATTA TTTAATGTGGTTGGATCCTCTTTGCAGGTC AACAAGGAGAACATG 1 (SEQ ID NO:263) (SEQ ID NO:264) (SEQID NO:265) (SEQ ID NO:266) 4 AGGTTTCAGCTTCTC ATTATTTAATGTGGTCCTCTCTCTCTGCCT CTCCTCTTTGCAGGT 3 (SEQ ID NO:267) (SEQ ID NO:268) (SEQID NO:269) (SEQ ID NO:270) 5 CTGACTGCTGTGCTC CAGGTTGCCACTGGACCCCACCACAGGCAG CTCAGATACACTTGG 3 (SEQ ID NO:271) (SEQ ID NO:272) (SEQID NO:273) (SEQ ID NO:274) 6 CCTCCAGGTAAGGTT TCAGCTTCTCATTATTGCTGTCTACTGATC GGGTAGACTACAAAG 0 (SEQ ID NO:275) (SEQ ID NO:276) (SEQID NO:277) (SEQ ID NO:278) 7 AGGTTTCAGCTTCTC ATTATTTAATGTGGTAAATGATAACCTCTC TCTCTGCCTCTCCTC 0 (SEQ ID NO:279) (SEQ ID NO:280) (SEQID NO:281) (SEQ ID NO:282) 8 GCTTGTTATGACCCC ACCACAGGCAGCTCAGACATGGAGAAGGCTC TGACCCCTGAGTTGC 7 (SEQ ID NO:283) (SEQ ID NO:284) (SEQID NO:285) (SEQ ID NO:286) 9 TTCAGCTTCTCATTA TTTAATGTGGTTGGACATTCTTTGAGACAC TAACGTGTCCCCAGG 0 (SEQ ID NO:287) (SEQ ID NO:288) (SEQID NO:289) (SEQ ID NO:290) 10 CTTGCTGACTGCTGT GCTCCAGGTTGCCACCTCTGACCCCTGAGT TGCTGTCTACTGATC 6 (SEQ ID NO:291) (SEQ ID NO:292) (SEQID NO:293) (SEQ ID NO:294)NOTESA: AG/GU CONSENSUS;B: IDENTICAL SEQUENCE;C: FROM TRANSFECTION/IVT EXPERIMENT;D: FROM TRANSLATION EXPERIMENTS

Three additional observations from these experiments are noteworthy.First, when non-neuronal cells from the same cultures are imaged todiscern RBP-GFP expression, a distinct staining pattern is routinelyobserved when compared to neurons that are transfected on the samecoverslip. For a majority of the RBP-GFP constructs shown here (with theexception of U2AF65-GFP and Magoh-GFP), subcellular distribution in gliais normally limited to nuclei with extremely modest, if any at all,staining visible in the cytoplasm (see FIG. 10 and FIGS. 2B-2E). Second,when neurons are transfected with the pEGFP-N1 or pDsRed-N1 constructalone, two types of expression are observed; neither of which are seenwhen we have fused a splice factor RBP in frame with GFP or DsRed. Inone instance, GFP is seen distributed throughout the neuronal nuclei andcytoplasm with equal intensity. Often it is not easy to discern aseparate nucleus in these GFP-expressing cells. In another instance, thenuclei of neurons and glia have low diffuse levels of GFP staining innuclei that is offset with distinct cytoplasmic staining and a visibleperinuclear ring of expression (FIGS. 10F and 10G). Finally, when weused a well-characterized histone 2B-YFP construct (Platani et al.,2002, Nat. Cell. Biol. 4:502-8) we observed a primarily nuclear patternof expression (FIG. 10J). These results suggest that specific splicingfactors from each of the spliceosome subcomplexes are distributed withinthe dendritic cytoplasm of neurons, have a variant expression innon-neuronal cells and may be capable of constitutively splicingpre-mRNAs into mature transcripts. Furthermore, experiments conductedwith the histone 2B-YFP fusion construct suggests that there is nointrinsic feature of the transfection techniques or the over-expressingGFP fusion constructs that would predispose our experiments to observinga dendritic localization.

Experimental Example 2 Amplification of Spliced CDC RNA from IsolatedDendrites.

Given the presence of splicing machinery in dendrites, the potential forRNA splicing to occur in neuronal dendrites was investigated. To performthese experiments, we utilized a pre-RNA splicing construct comprised ofexons 14 and 15 of the CDC gene (Ohno et al., 1987, Proc. Natl. Acad.Sci. U.S.A. 84:5187-91 [38]) to assess whether it can be spliced inisolated dendrites.

During the time course of these experiments, the viability of isolateddendrites to splice pre-RNA transcripts was assesed. Isolated dendriteshave been previously shown to be translationally active (Aakalu et al.,2001, Neuron 30:489-502; Crino et al., 1996, Neuron 17:1173-87; Job etal., 2001, Proc. Natl. Acad. Sci. U.S.A 98:13037-42) with the additionalability to incorporate post-translational sugar precursors of thesecreted-protein protein-glycosylation pathway (Torre et al., 1996, J.Neurosci. 16:5967-78). One very sensitive measure of dendriticcompromise is mitochondrial function. JC-1, a mitochondrial membranepotential dye (Li et al., 2004, Cell 119:873-87; Arancia et al., 2004,Amino Acids 26:273-82; Ogbourne et al., 2004, Cancer Res. 64:2833-9),was added at varying time point to isolated dendrites. Sequestration ofJC-1 by functional mitochondria results in its polymerization and theformation of fluorescent red aggregates (see FIGS. 3 and 10). Acomparison of the zero time point and the 5 hr time point show similarlevels of red fluorescence. Based on these criteria, these isolateddendrites remain biochemically functional during the time frame of theseexperiments.

In the CDC pre-RNA the 87 bp exon 14 and 73 bp exon 15 of the chicken8-crystalline gene are interrupted by a 257 bp intron. The pre-RNAderived from this construct has been used for in vitro pre-RNA splicingassays where spliceosome assembly and CDC pre-RNA splicing were observed(Kataoka et al., 2000, Mol. Cell 6:673-82; Ohno et al., 1987, Proc.Natl. Acad. Sci. U.S.A. 84:5187-91). Briefly, CDC pre-RNA wastranscribed from the construct, encoated with the polycationic lipidMetafectene, and manually applied onto dendrites that were isolated fromtheir cognate cell somas as performed previously (Aakalu et al., 2001,Neuron 30:489-502; Crino et al., 1996, Neuron 17:1173-87; Job et al.,2001, Proc. Natl. Acad. Sci. U.S.A 98:13037-42; Kacharmina et al., 2000,Proc. Natl. Acad. Sci. U.S.A. 97:11545-50). After incubation at 37° C.for 30 minutes, RNA was extracted from these isolated dendrites. Reversetranscription of this RNA was primed with an SP6 directed primerinherent to the transfected 3′-end of the CDC RNA construct. The cDNAwas then used as template in multiple rounds of PCR using CDC-specificprimers with nested primer sets. These PCR amplicons were subcloned andsequenced to determine the splice boundaries utilized in the dendriticsubdomain of the neuron.

In addition to a large amount of unspliced CDC pre-RNA, sequenceanalysis of 53 spliced CDC RNA sequences isolated from eight independentexperiments revealed that CDC pre-RNA was spliced using both predicted,consensus and non-canonical splicing sequences (FIG. 2). Four of thesesequences adhere to the AG/GU rule for nuclear splicing, while the othersequences make use of cryptic splice sites (Lee et al. 1997, Cancer Res.57:3131-4). Four other sequences adhere to atypical AT-AC intronsplicing, an event requiring the incorporation of SR proteins (Hastingset al., 2001, Rna 7:471-82). Approximately 50% of successfultransfection assays resulted in the production of spliced mRNAssuggesting that a subset of dendrites capable of splicing. As with anyPCR-dependent protocol, aberrant amplification of unspliced CDC pre-RNAor spliced CDC RNA through mispriming could result in a truncated DNAfragment that would mimic RNA splicing. If these data were the result ofsuch mispriming (internal priming of the CDC cDNA at regions of homologyin other CDC cDNAs), then we would expect that a majority of transcriptscontain three or more bases of similarity in the donor and acceptorsites at the splice junction (the number of 3′-end primer matched basesto prime PCR) (Wu et al., 1991, DNA Cell Biol. 10:233-8; Liang et al.,1995, Curr. Opin. Immunol. 7:274-80). In contrast, ⅔ of the splicedsequences have two or less bases overlap, suggesting that the PCRreaction was of high fidelity. Furthermore, controls in all experimentsshowed that PCR amplification of the CDC pre-RNA does not give rise toamplicons distinct from the CDC pre-RNA template.

To show that this splicing event wasn't unique to the CDC RNA,experiments were repeated using a second, independent constructretaining the SV40 small t-antigen intron. The pGL-2 splicing construct(Promega, Carlsbad, Calif.) contains the luciferase coding region fusedto the SV40 small t-antigen intron upstream of the luciferasepolyadenylation site. This construct has been used extensively insplicing experiments as a control where it is efficiently spliced inmammalian cells. This luciferase-SV40 sequence was PCR amplified fromthe pGL-2 plasmid with the 5′-luciferase directed primer containing a T7RNA polymerase promoter site so that sense RNA can be made from thisconstruct. Following transfection splice product sequences from theluciferase-SV40 chimera were analyzed as described for the CDC pre-RNAsplicing experiments using specific primers directed against bases1680-1700, 1700-1720,2580-2600, and 2680-2700. Five unique splicedsequences were detected (FIG. 1). As observed with CDC pre-RNA, bothconventional and cryptic splice donor/acceptor sites are observed.Cryptic splicing sites with the SV40 small t-antigen intron 3′ to somegenes has been previously observed (Evans et al., 1989, Gene 84:135-42;Huang et al., 1990, Mol. Cell Biol. 10:1805-10). The characterizedsequences are spliced forms some of which show only two bases of‘overlap’ as per the pre-CDC splicing discussion above. These dataconfirm and extend the data from the CDC RNA transfection studies, andfurther show the RNA splicing capacity of dendrites.

Amplification of spliced CDC pre-RNA from isolated, Sm antigen-positivedendrites. To conclusively show that a dendrite containing an identifiedsplicing factor can also splice pre-RNA, intact neurons were transfectedwith CDC pre-RNA, fixed at a later time point with 4% paraformaldehyde,and the dendritically-localized CDC RNA was copied into cDNA using anSP6 primer in situ (FIG. 5A). These dendrites were thenimmunohistochemically stained for Sm proteins, and a single processcontaining granule-like structures were harvested. The punctate stainingseen in FIG. 5 is qualitatively similar to that seen previously (FIG.2E). The in situ transcribed cDNA was isolated and used as a PCRtemplate for amplification of spliced CDC RNA. The amplicons of splicedCDC RNA migrates faster than non-spliced CDC pre-RNA during agarose gelelectrophores (FIG. 5B) and shows splice donor/acceptor sequencesimilarity to previous experiments (FIGS. 1 and 5). These data show thatSm antigen-positive processes are capable of supporting dendriticpre-RNA splicing. In experiments where Sm antigen was absent fromdendrites, PCR amplification of CDC pre-RNA transfected, Sm-negative,processes yielded no spliced RNAs, suggesting that the inclusion of Smantigen in dendrites is correlated with the functional splicing activityin dendrites.

Experimental Example 3 Synaptoneurosomes can Splice CDC pre-RNA.

Synaptoneurosomes have been used to demonstrate protein synthesis indendritically enriched regions of neurons (Weiler et al., 1993, Proc.Natl. Acad. Sci. U.S.A. 90:7168-71; Rao et al., 1993, J. Neurochem.61:835-44; Bagni et al., 2000, J. Neurosci. 20:RC76). This subcellularfraction of tissue homogenates contains liposomes of pre- andpost-synaptic entities. Visually, these fractions are absent nuclei(Weiler et al., 1993, Proc. Natl. Acad. Sci. U.S.A. 90:7168-71; Rao etal., 1993, J. Neurochem. 61:835-44; Bagni et al., 2000, J. Neurosci.20:RC76). Using established protocols, we isolated freshsynaptoneurosomes from rat brain and tested them for the presence ofsplicing proteins using western blotting. As seen in FIG. 6, U2AF65,pan-SR, SC-35, SF2 and Sm antigens were all present in the whole brain(WB) extract as well as in the synaptoneurosome (SN) preparation. Theseresults show that these same pre-spliceosome proteins, localized incultured primary dendrites, are present in the WB and, more importantly,the SN fraction at differing abundances. In this respect, the westernanalysis data is consistent with the immunofluorescence data of FIG. 2.While SN preparations are never purely neuronalin origin, it is unlikelythat the observed differing intensities of the protein bands observedwith chemiluminscence is function of nuclear contamination as thesepreparations contain virtually no nuclei and less than 5% mitochondria(Booth et al., 1978, Biochem. J. 176:365-70). Tubulin was used as aloading control to show that the same amount of protein was loaded inthe different lanes.

Given the presence of these proteins in SNs, the SN extracts were testedfor the ability to splice the CDC pre-RNA. Freshly prepared SNs weresupplemented with 1.5 mM ATP and incubated with CDC pre-RNA for 30minutes at 37° C. After incubation, RNA was isolated from the SNs andthe spliced CDC RNA was PCR amplified, cloned and sequenced. Ten splicedsequences generated from three separate experiments are shown in FIG. 1.Consistent with the ATP-dependent nature of the spliceosome and therelative absence of mitochondria in these fractions, no splice formswere generated when ATP was omitted from the SN preparation. A similareffect was observed when SNs were subjected to a single freeze/thawcycle prior to the splicing assay.

Experimental Example 4 Spliced CDC RNA can be Translated in IsolatedDendrites.

As described previously, the isolated dendrite assay involves mechanicalsevering of the dendrites from the cell soma, removal of the soma, andtransfection of the isolated dendrites. FIGS. 7A, 7D and 7G showphotomicrographs of primary rat hippocampal neurons with the cell somasintact (white arrows). FIGS. 7B, 7E and 7H show these same microscopicfields after removal of the soma, leaving the dendrites (black arrows).Since the CDC pre-RNA contains an in-frame FLAG epitope-tag in thesecond exon this epitope should be immunohistochemically detectable ifthe dendritically spliced CDC pre-RNA can be translated. Unspliced CDCpre-RNA will not produce a translated FLAG epitope. These same dendritesare stained for FLAG expression after mock transfection (Panel C) andtransfection with mature CDC RNA (Panel F) or unspliced CDC pre-RNA(Panel I). These data show the similarity in FLAG sequence expressionlevels in dendrites when transfected with mature CDC RNA and CDCpre-RNA.

Dendritic transfection of CDC pre-RNA can result in the isolation ofalternatively spliced CDCRNAs that contain two adjacent open readingframes (ORFs), with the 3′ ORF containing the FLAG epitope (FIG. 1).Therefore, as a result of splicing, bicistronic mRNAs were created fromthe CDC pre-RNA. To assess whether the FLAG-tags on downstream ORFs aretranslatable, the 236 bp (splice variant 1), 181 bp (splice variant 6),and 146 bp (splice variant 45) cDNAs were selected and transcribed intoRNA that was then transfected into the isolated dendrites, followed byimmunostaining with the anti-FLAG antibody. The sequence of the 236 bp,181 bp, and 146 bp cDNAs suggests that if the consensus splice site isnot utilized, the sequence extension of the 3′-end of the first exonwill give rise to an in-frame termination codon producing a smalltranslational unit. This 5′ translational unit does not contain theFLAG-tag. The 236 and 181 bp clones both contain a second open readingframe that, if translated, would contain the FLAG-tag. The 146 basesequence is spliced so that there is no in-frame initiator methioninethat could prime the expression of the FLAG-tag. FIG. 8 shows that uponin vivo translation, both the 236 bp and 181 bp alternatively splicedCDC RNAs gave rise to FLAG antigenicity (FIGS. 8A-8B and 8C-8D,respectively). Transfection of the 146 bp RNA did not produce FLAGcontaining protein (FIGS. 8E and 8F). These data indicate that 1) thein-frame methionine for the predicted second open-reading frame producedby nontraditional splicing of the CDC pre-RNA can be recognized by thedendritic protein synthesis machinery and utilized as an initiatormethionine to produce protein from the second open-reading frame and 2)these transfected spliced RNAs can be further spliced in the dendrite toyield a mature CDC RNA. The translation of the second open-reading frameof a bicistronic mRNA could occur by read-through of the second readingframe as the ribosomes that translated the first open-reading frame movealong the RNA after termination of translation of the first open-readingframe (Kozaket et al., 1998, Nucleic Acids Res. 26:4853-9).Alternatively, translation could occur through utilization of aninternal ribosome entry site (IRES) that may be present in theinter-cistronic region in a cap-independent process (Macejak et al.,1991, Nature 353:90-4 [54]). M-fold analysis of the spliced CDC RNAsreveals stem-loops and complex secondary structures that have beenimplicated as potential IRES sites (Martinez-Salas et al., 2002,Biochimie 84:755-63). Regardless of translational mechanism, these datashow that dendritically spliced RNAs can be translated in the localdendritic environment.

Experimental Example 5 Control Data for GFP Fusion Constructs

Control experiments were conducted using the Olympus Fluoview FV100spectrometer to illustrate the specificity of the GFP fusion constructsused in FIG. 4. As noted elsewhere herein, the intensity of the laserwas increased to facilitate imaging of dendritic fluorescence. Thefluorescence of the nuclei in these experiments was saturated. In FIG.10A, a representative image of the “speckled” nuclear pattern ofexpression is shown using the UAP56-GFP construct. Similar patterns ofsignal were observed for all GFP fusion proteins exemplified herein. Theimages set forth in FIG. 10 have been optimized for nuclear images sothe intensity of the laser is very low with no increase in gain. Aghosting of GFP expression is visible in the perinuclear region and ifthe intensity of the laser was increased dendritic staining would bevisible as well. In FIGS. 10B-10E are illustrated the two types ofnon-neuronal staining that observed with GFP fusion constructs set forthin detail elsewhere herein. In the first example, here exemplified bySF1/mBBP-GFP (FIG. 10B) and its corresponding MAP2 immunofluorescence(FIG. 10C), expression is strongly localized to the nucleus with verylow levels of cytoplasmic signal. This type of staining pattern wasobserved for UAP56-GFP and Y14-GFP as well. A second non-neuronalRBP-GFP phenotype is visualized in FIG. 10D with the U2AF65-GFPconstruct, in which can be observed a well-defined expression in thenucleus with low, but significant fluorescence visible throughout theglial cytoplasm. A similar staining pattern is observed for Magoh-GFP inthe glial cell of these cultures (see also FIG. 4A). In FIG. 10E, atransfected neuron (note the MAP2 fluorescence) is also visible in thelower left quadrant of the image. The intensity of staining is apparentin the neuron versus the glia. This difference in RBP-GFP expression wastypical when comparing basal levels of expression in neurons and glia.Finally, the expression of the plain pEGFP-N1 construct is illustratedwhen expressed in glia (FIG. 10F) or neurons (FIG. 10G). There is anobvious difference in the patterns for non-neuronal cells when comparingFIG. 10F to FIGS. 10B and 10D. Similary, when GFP expression is neuronsis not fused to a RBP as described in the present disclosure, there arenot distinct patterns of expression. GFP is seen diffusely spread overthe nucleus and cytoplasm. This is in contrast to the patterns ofexpression seen in FIGS. 2 and 4 illustrating the localization of thenative protein or the expression of a GFP fusion protein. Finally, inFIGS. 10E-10H aer shown control experiments with antigens that shouldretain a predominantly nuclear localization. An anti-histone 3 antibody(E) and a histone 2B-YFP fusion construct (G) were used to show therespective neuronal localization. Corresponding phase-contrast photos(FIGS. 10F and 10H) illustrate the neuronal morphology. These data showthat the use of overexpressing GFP or YFP constructs do not, in allcircumstances, lead to localization in the dendritic fields. The arrowsin FIGS. 10E and 10F show where the dendrites are localized in theimages, while the arrows in FIG. 10G and 10H show the dendrites in thisimage.

Experimental Example 6 Isolated Dendrite Viability Evaluation usingMitochondrial Function Measurement

Dendrites were isolated from cortical cell cultures and at various timesafter severing, JC-1 dye (Molecular Probes) was added at a concentrationof 1 μg dye/ml media. The dye was incubated at 37° C. with the culturesfor 10 min followed by imaging with fluorescence microscopy. Functionalmitochondria take up and concentrate the green fluorescent monomer. Oncethe concentration reached a threshold, the dye began to polymerize,forming red fluorescent aggregates visualized as red puncta. Acomparison of the zero time point and the 5 hour time point show similarlevels of red fluorescence (FIG. 11).

The disclosures of each and every patent, patent application, andpublication cited herein are hereby incorporated herein by reference intheir entirety.

While this invention has been disclosed with reference to specificembodiments, it is apparent that other embodiments and variations ofthis invention may be devised by others skilled in the art withoutdeparting from the true spirit and scope of the invention. The appendedclaims are intended to be construed to include all such embodiments andequivalent variations.

1. A method of remodeling a dendrite, said method comprising the stepsof: a. transfecting a dendrite with an RNA comprising at least oneintron, wherein said dendrite comprises at least one component of aspliceosome and further wherein said component of a spliceosome iscapable of splicing an RNA; b. allowing said RNA comprising at least oneintron to be spliced by said spliceosome components; and c. allowingsaid spliced RNA to be translated in said dendrite; wherein saiddendrite is thereby remodeled as a consequence of said translation.
 2. Amethod of remodeling a dendrite interaction, said method comprising thesteps of: a. transfecting a dendrite with an RNA comprising at least oneintron, wherein said dendrite comprises at least one component of aspliceosome and further wherein said component of a spliceosome iscapable of splicing an RNA; b. allowing said RNA comprising at least oneintron to be spliced by said spliceosome components; and c. allowingsaid spliced RNA to be translated in said dendrite; wherein saiddendrite interaction is thereby remodeled as a consequence of saidtranslation.
 3. A method of remodeling a synaptic network comprisinginteraction with at least one dendrite, said method comprising the stepsof: a. transfecting a dendrite with an RNA comprising at least oneintron, wherein said dendrite comprises at least one component of aspliceosome and further wherein said component of a spliceosome iscapable of splicing an RNA; b. allowing said RNA comprising at least oneintron to be spliced by said spliceosome components; and c. allowingsaid spliced RNA to be translated in said dendrite; wherein saidsynaptic network is thereby remodeled as a consequence of saidtranslation.
 4. A method of splicing an RNA, said method comprising thesteps of: a. providing an isolated dendrite comprising at least onecomponent of a spliceosome, wherein said component is capable ofsplicing an RNA; and b. transfecting said dendrite with an RNAcomprising at least one intron; wherein said RNA comprising at least oneintron is spliced by said spliceosome components.
 5. The method of claim1, wherein said dendrite is a component of a neuron.
 6. The method ofclaim 1, wherein said dendrite is an isolated dendrite.
 7. A method ofsplicing an RNA, said method comprising the steps of: a. providing anisolated cell comprising at least one component of a spliceosome in thecytoplasm, wherein said component is capable of splicing an RNA; and b.transfecting said cell with an RNA comprising at least one intron;wherein said RNA comprising at least one intron is spliced by saidspliceosome components in said cytoplasm.
 8. The method of claim 1,wherein RNA splicing donor/acceptor pairs are selected from the groupconsisting of canonical, atypical and cryptic.
 9. The method of claim 1,wherein said RNA comprising at least one intron is a pre-RNA.
 10. Themethod of claim 9, wherein said pre-RNA is a pre-mRNA.
 11. The method ofclaim 1, wherein said RNA comprising at least one intron is derived froma nucleic acid comprising a construct selected from the group consistingof pEGFP-N1, pDsRed-N1, SF1/mBBP-GFP, U2AF65-GFP, a GFP construct, aDsRed construct, a histone 2B-YFP construct.
 12. The method of claim 1,wherein at least one spliceosome component is selected from the groupconsisting of Y14, Magoh, RNPS 1, SC-35, SF2, U2AF65, Smith antigen,pan-SR antigen, U1 snRNP, U2 snRNP, U4 snRNP, U5 snRNP, U6 snRNP.
 13. Amethod of translating an RNA, said method comprising the steps of: a.providing an isolated dendrite comprising at least one component of aspliceosome, wherein said component is capable of splicing an RNA; andb. transfecting said dendrite with an RNA comprising at least oneintron, wherein said RNA is spliced by said spliceosome components;further wherein said spliced RNA is translated.
 14. A method oftranslating an RNA, said method comprising the steps of: a. providing anisolated cell comprising at least one component of a spliceosome in thecytoplasm, wherein said component is capable of splicing an RNA; and b.transfecting said cell with an RNA comprising at least one intron,wherein said RNA is spliced by said spliceosome components in saidcytoplasm; further wherein said spliced RNA is translated.
 15. A methodof splicing an RNA, said method comprising the steps of: a. providing anisolated synaptoneurosome comprising at least one component of aspliceosome, wherein said component is capable of splicing an RNA; andb. contacting said synaptoneurosome with an RNA comprising at least oneintron; wherein said RNA comprising at least one intron is spliced bysaid spliceosome components.
 16. A method of translating an RNA, saidmethod comprising the steps of: a. providing an isolatedsynaptoneurosome comprising at least one component of a spliceosome,wherein said component is capable of splicing an RNA; b. contacting saidsynaptoneurosome with an RNA comprising at least one intron, whereinsaid RNA is spliced by said spliceosome components; and c. contactingsaid spliced RNA with a composition capable of translating an RNA underconditions suitable for translating an RNA.